ez dna methylation gold kit  (Zymo Research)


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    Name:
    EZ DNA Methylation Gold Kit
    Description:
    The EZ DNA Methylation Gold Kit is a refinement of our popular EZ DNA Methylation kit and uses heat denaturation instead of chemical denaturation of the input DNA This allows for the denaturation and bisulfite conversion steps to be consolidated into one step leading to a much reduced incubation time Recovered bisulfite converted DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion sequencing microarrays etc
    Catalog Number:
    d5006
    Price:
    None
    Applications:
    Bisulfite Conversion
    Size:
    50 units
    Category:
    Life Science Reagents and Media
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    Structured Review

    Zymo Research ez dna methylation gold kit
    EZ DNA Methylation Gold Kit
    The EZ DNA Methylation Gold Kit is a refinement of our popular EZ DNA Methylation kit and uses heat denaturation instead of chemical denaturation of the input DNA This allows for the denaturation and bisulfite conversion steps to be consolidated into one step leading to a much reduced incubation time Recovered bisulfite converted DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion sequencing microarrays etc
    https://www.bioz.com/result/ez dna methylation gold kit/product/Zymo Research
    Average 95 stars, based on 2135 article reviews
    Price from $9.99 to $1999.99
    ez dna methylation gold kit - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency"

    Article Title: Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2016.08.013

    DNA Methylation Differences Observed in MLL-AF4 + B Cell Blasts Versus CD34 + Cells Expressing MLL-AF4 and CD34 + CD19 + B Cell HPCs (A) Global DNA methylation analysis by pyrosequencing of LINE-1 elements in MLL-AF4 + blasts and normal CD34 + CD19 + B cell HPCs (n = 3 independent experiments). Error bars indicate SD. (B) Unsupervised hierarchical clustering and heatmap showing the CpG sites with differential DNA methylation between MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + cells expressing ectopic MLL-AF4. Average methylation values are displayed from 0 (blue) to 1 (yellow). (C) Venn diagrams showing the number of CpG sites differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. (D) Selection of GO terms from the top 50 statistically significant biological functions, ranked by p value (x axis), of genes differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. The y axis indicates the relative risk (±95% confidence intervals) as a measure of effect size. The relative risk is the ratio of the proportion of genes belonging to a given GO term in a selected subset of genes to the same proportion in the remaining, background genes.
    Figure Legend Snippet: DNA Methylation Differences Observed in MLL-AF4 + B Cell Blasts Versus CD34 + Cells Expressing MLL-AF4 and CD34 + CD19 + B Cell HPCs (A) Global DNA methylation analysis by pyrosequencing of LINE-1 elements in MLL-AF4 + blasts and normal CD34 + CD19 + B cell HPCs (n = 3 independent experiments). Error bars indicate SD. (B) Unsupervised hierarchical clustering and heatmap showing the CpG sites with differential DNA methylation between MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + cells expressing ectopic MLL-AF4. Average methylation values are displayed from 0 (blue) to 1 (yellow). (C) Venn diagrams showing the number of CpG sites differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. (D) Selection of GO terms from the top 50 statistically significant biological functions, ranked by p value (x axis), of genes differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. The y axis indicates the relative risk (±95% confidence intervals) as a measure of effect size. The relative risk is the ratio of the proportion of genes belonging to a given GO term in a selected subset of genes to the same proportion in the remaining, background genes.

    Techniques Used: DNA Methylation Assay, Expressing, Methylation, Selection

    2) Product Images from "SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation"

    Article Title: SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation

    Journal: Nature

    doi: 10.1038/nature12931

    Tethered SUVH2 recruits Pol V through DRD1, resulting in DNA methylation and a late-flowering phenotype a. Plants grown side-by-side to illustrate early flowering of ZF-SUVH2 in fwa-4 (T2 plants) compared to fwa-4. b. Flowering time of Columbia-0 (WT), ZF-SUVH2 in fwa-4 , ZF-KYP in fwa-4 , HA-SUVH2 in fwa-4 , and fwa-4 . Flowering time was determined by counting all rosette and cauline leaves up until the terminal flower. The average leaf number and standard deviation of between 20-30 plants was determined. Mean +/− SD. c. Percent methylation at each cytosine in the FWA repeat region as determined by BS-seq in T2 and T3 ZF-SUVH2/ fwa-4 plants compared to T2 ZF-KYP/ fwa-4 (unmethylated) and WT (standard methylation pattern). ZF binding sites are shown in green and the FWA gene in blue. d. NRPE1 ChIP in WT (positive control), nrpe1 mutant (negative control), fwa-4 epiallele, and ZF-SUVH2/ fwa-4 . qPCR results of two well-characterized NRPE1 binding sites (IGN5 and IGN22) and two regions in FWA (FWAp: promoter; FWAt: transcript) are shown as enrichment of IP/input relative to negative control. Mean +/− SD of two biological replicas. e. Coimmunoprecipitation of HA-SUVH2 in Arabidopsis using Flag-DRD1. Left panels are inputs from the two parental strains (expressing either HA-SUVH2 (HA-2) or Flag-DRD1 (Flag-D)) and an F2 line expressing both HA-SUVH2 and Flag-DRD1 (HA-2xFlag-d). The right panels show elution from Flag-magnetic beads. Top panels are HA western blots, bottom panels are Flag western blots.
    Figure Legend Snippet: Tethered SUVH2 recruits Pol V through DRD1, resulting in DNA methylation and a late-flowering phenotype a. Plants grown side-by-side to illustrate early flowering of ZF-SUVH2 in fwa-4 (T2 plants) compared to fwa-4. b. Flowering time of Columbia-0 (WT), ZF-SUVH2 in fwa-4 , ZF-KYP in fwa-4 , HA-SUVH2 in fwa-4 , and fwa-4 . Flowering time was determined by counting all rosette and cauline leaves up until the terminal flower. The average leaf number and standard deviation of between 20-30 plants was determined. Mean +/− SD. c. Percent methylation at each cytosine in the FWA repeat region as determined by BS-seq in T2 and T3 ZF-SUVH2/ fwa-4 plants compared to T2 ZF-KYP/ fwa-4 (unmethylated) and WT (standard methylation pattern). ZF binding sites are shown in green and the FWA gene in blue. d. NRPE1 ChIP in WT (positive control), nrpe1 mutant (negative control), fwa-4 epiallele, and ZF-SUVH2/ fwa-4 . qPCR results of two well-characterized NRPE1 binding sites (IGN5 and IGN22) and two regions in FWA (FWAp: promoter; FWAt: transcript) are shown as enrichment of IP/input relative to negative control. Mean +/− SD of two biological replicas. e. Coimmunoprecipitation of HA-SUVH2 in Arabidopsis using Flag-DRD1. Left panels are inputs from the two parental strains (expressing either HA-SUVH2 (HA-2) or Flag-DRD1 (Flag-D)) and an F2 line expressing both HA-SUVH2 and Flag-DRD1 (HA-2xFlag-d). The right panels show elution from Flag-magnetic beads. Top panels are HA western blots, bottom panels are Flag western blots.

    Techniques Used: DNA Methylation Assay, Standard Deviation, Methylation, Binding Assay, Chromatin Immunoprecipitation, Positive Control, Mutagenesis, Negative Control, Real-time Polymerase Chain Reaction, Expressing, Magnetic Beads, Western Blot

    ZF-SUVH2 construct stably recruits Pol V to FWA through a direct interaction with DRD1 a. Top: Diagram of SUVH2 with Zn Finger (ZF) inserted immediately before the HA tag. Bottom: Schematic of FWA gene showing the two small and two large repeats (blue arrows), the regions amplified by PCR (promoter and transcript: green lines), the start and direction of transcription (red arrow), and the sites bound by the ZF (indicated by two orange arrows). b. Flag-ChIP in WT versus ZF-KYP (flag-tagged) showing enrichment at FWA in both the promoter and transcript region (see above). c. Percent methylation at each C in the FWA repeat region from three individual T1 plants. Percent methylation was determined from 20-25 clones of bisulfite-treated DNA. d. BS-Seq of FWA from a Basta-resistant line containing the ZF-SUVH2 transgene and two Basta-sensitivie siblings which had lost the ZF-SUVH2 transgene. e. Pull-down of DRD1-Flag with ZF-SUVH2. Input: DRD1-Flag extract from Arabidopsis; Beads-mock: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana extract; Beads-ZF-SUVH2: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana ZF-SUVH2 extract. Top panel: Flag blot; bottom panel: HA blot.
    Figure Legend Snippet: ZF-SUVH2 construct stably recruits Pol V to FWA through a direct interaction with DRD1 a. Top: Diagram of SUVH2 with Zn Finger (ZF) inserted immediately before the HA tag. Bottom: Schematic of FWA gene showing the two small and two large repeats (blue arrows), the regions amplified by PCR (promoter and transcript: green lines), the start and direction of transcription (red arrow), and the sites bound by the ZF (indicated by two orange arrows). b. Flag-ChIP in WT versus ZF-KYP (flag-tagged) showing enrichment at FWA in both the promoter and transcript region (see above). c. Percent methylation at each C in the FWA repeat region from three individual T1 plants. Percent methylation was determined from 20-25 clones of bisulfite-treated DNA. d. BS-Seq of FWA from a Basta-resistant line containing the ZF-SUVH2 transgene and two Basta-sensitivie siblings which had lost the ZF-SUVH2 transgene. e. Pull-down of DRD1-Flag with ZF-SUVH2. Input: DRD1-Flag extract from Arabidopsis; Beads-mock: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana extract; Beads-ZF-SUVH2: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana ZF-SUVH2 extract. Top panel: Flag blot; bottom panel: HA blot.

    Techniques Used: Construct, Stable Transfection, Amplification, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Methylation, Clone Assay, Magnetic Beads

    3) Product Images from "A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation"

    Article Title: A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation

    Journal: Genome Medicine

    doi: 10.1186/s13073-015-0246-z

    Validation of top-ranked DMP cg20475486 using a clinical biomarker assay. Replication of the top-ranked DMP associated with aGVHD severity, cg20475486, using a semi-quantitative DNA methylation assay. a Box-and-whisker plot of DNA methylation values in graft donors in T cell-depleted HSCT (initial discovery cohort). We replicated the DNA hypomethylation phenotype in HSCT donors matched to recipients with severe aGVHD compared to no/mild aGVHD ( P = 0.039, Wilcoxon rank-sum test). b At a relative DNA methylation threshold of 8.295 (dotted line), the AUC was 0.74 with a maximal specificity and sensitivity of 0.75 and 0.71, respectively. c Box-and-whisker plot of DNA methylation values in graft donors in T cell-replete HSCT (that is, without the application of in vivo alemtuzumab). In an independent sample cohort, we confirmed the observed DNA methylation phenotype, suggesting the epigenetic classifier is also effective in the context of a T cell-replete conditioning regimen ( P = 0.050). For two samples, C t -values could not be detected in the MethyLight experiments. d At a threshold of PMR = 17.73 (dotted line), the area under the ROC curve was 0.73 with a maximal specificity and sensitivity of 0.71 and 0.78, respectively
    Figure Legend Snippet: Validation of top-ranked DMP cg20475486 using a clinical biomarker assay. Replication of the top-ranked DMP associated with aGVHD severity, cg20475486, using a semi-quantitative DNA methylation assay. a Box-and-whisker plot of DNA methylation values in graft donors in T cell-depleted HSCT (initial discovery cohort). We replicated the DNA hypomethylation phenotype in HSCT donors matched to recipients with severe aGVHD compared to no/mild aGVHD ( P = 0.039, Wilcoxon rank-sum test). b At a relative DNA methylation threshold of 8.295 (dotted line), the AUC was 0.74 with a maximal specificity and sensitivity of 0.75 and 0.71, respectively. c Box-and-whisker plot of DNA methylation values in graft donors in T cell-replete HSCT (that is, without the application of in vivo alemtuzumab). In an independent sample cohort, we confirmed the observed DNA methylation phenotype, suggesting the epigenetic classifier is also effective in the context of a T cell-replete conditioning regimen ( P = 0.050). For two samples, C t -values could not be detected in the MethyLight experiments. d At a threshold of PMR = 17.73 (dotted line), the area under the ROC curve was 0.73 with a maximal specificity and sensitivity of 0.71 and 0.78, respectively

    Techniques Used: Biomarker Assay, DNA Methylation Assay, Whisker Assay, In Vivo

    Overview of the study design. We aimed to identify specific epigenetic marks in peripheral blood of healthy graft donors that delineate aGVHD severity in HLA-matched sibling recipients prior to HSCT. At the discovery stage, we assessed genome-wide DNA methylation levels in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, that is, ‘severe’ aGVHD (grades III + IV; n = 9) and ‘no/mild’ aGVHD (grades 0, I + II; n = 76). HSCT recipients received reduced-intensity (non-myeloablative) T cell-depleted conditioning using in vivo alemtuzumab. At the replication stage, we used a semi-quantitative DNA methylation assay, MethyLight, which can be easily used in a clinical setting. We validated the top-ranked differentially methylated positions associated with aGVHD severity status in donors in the context of both T cell-depleted and T cell-replete conditioning regimens for HSCT
    Figure Legend Snippet: Overview of the study design. We aimed to identify specific epigenetic marks in peripheral blood of healthy graft donors that delineate aGVHD severity in HLA-matched sibling recipients prior to HSCT. At the discovery stage, we assessed genome-wide DNA methylation levels in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, that is, ‘severe’ aGVHD (grades III + IV; n = 9) and ‘no/mild’ aGVHD (grades 0, I + II; n = 76). HSCT recipients received reduced-intensity (non-myeloablative) T cell-depleted conditioning using in vivo alemtuzumab. At the replication stage, we used a semi-quantitative DNA methylation assay, MethyLight, which can be easily used in a clinical setting. We validated the top-ranked differentially methylated positions associated with aGVHD severity status in donors in the context of both T cell-depleted and T cell-replete conditioning regimens for HSCT

    Techniques Used: Genome Wide, DNA Methylation Assay, In Vivo, Methylation

    4) Product Images from "Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat"

    Article Title: Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat

    Journal: Journal of Neuroendocrinology

    doi: 10.1111/jne.12371

    Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P
    Figure Legend Snippet: Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P

    Techniques Used: Methylation, Clone Assay

    5) Product Images from "Gestational Choline Supply Regulates Methylation of Histone H3, Expression of Histone Methyltransferases G9a (Kmt1c) and Suv39h1 (Kmt1a), and DNA Methylation of Their Genes in Rat Fetal Liver and Brain *"

    Article Title: Gestational Choline Supply Regulates Methylation of Histone H3, Expression of Histone Methyltransferases G9a (Kmt1c) and Suv39h1 (Kmt1a), and DNA Methylation of Their Genes in Rat Fetal Liver and Brain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807651200

    Methylation-specific PCR ( MSP ) analysis of G9a and Suv39h1 promoter CpG islands. A–C , genomic DNA isolated from E17 liver ( A and B ) or cortex ( C ) was treated with sodium bisulfite and analyzed by methylation-specific PCR. The inset shows examples
    Figure Legend Snippet: Methylation-specific PCR ( MSP ) analysis of G9a and Suv39h1 promoter CpG islands. A–C , genomic DNA isolated from E17 liver ( A and B ) or cortex ( C ) was treated with sodium bisulfite and analyzed by methylation-specific PCR. The inset shows examples

    Techniques Used: Methylation, Polymerase Chain Reaction, Isolation

    6) Product Images from "Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma"

    Article Title: Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055761

    Genome-wide Methylation450 BeadChip DNA methylation portrait. A. Left: Significant differential methylation (DM) among the 485,577 CpG loci with a cut-off as Bonferroni adjusted p-value
    Figure Legend Snippet: Genome-wide Methylation450 BeadChip DNA methylation portrait. A. Left: Significant differential methylation (DM) among the 485,577 CpG loci with a cut-off as Bonferroni adjusted p-value

    Techniques Used: Genome Wide, DNA Methylation Assay, Methylation

    7) Product Images from "Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line"

    Article Title: Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20144058

    XIST expression. A , Relative expression levels of XIST RNA in HCT116 and a female cell line. The expression of YWAHZ was used as a reference. B , XIST RNA FISH in female cell line ( i ) and male HCT116 cell line treated with 10 µM 5-aza-CdR (5-aza-2′-deoxycytidine) for 96 h ( ii ). Nuclei were counterstained with DAPI (blue) and XIST RNA signals are red. The scale bar corresponds to 10 µm. C , XIST DNA methylation pattern by 8 CpGs (cytosine-phosphate-guanine) sites of 450K platform (cg15319295, cg12653510, cg05533223, cg117117280, cg20698282, cg17513789, cg02644889, and cg17279685). The color-ratio bar at the bottom indicates the methylation level. D , DNA methylation level of CpG sites related to X chromosome covered in the 450K platform; **P
    Figure Legend Snippet: XIST expression. A , Relative expression levels of XIST RNA in HCT116 and a female cell line. The expression of YWAHZ was used as a reference. B , XIST RNA FISH in female cell line ( i ) and male HCT116 cell line treated with 10 µM 5-aza-CdR (5-aza-2′-deoxycytidine) for 96 h ( ii ). Nuclei were counterstained with DAPI (blue) and XIST RNA signals are red. The scale bar corresponds to 10 µm. C , XIST DNA methylation pattern by 8 CpGs (cytosine-phosphate-guanine) sites of 450K platform (cg15319295, cg12653510, cg05533223, cg117117280, cg20698282, cg17513789, cg02644889, and cg17279685). The color-ratio bar at the bottom indicates the methylation level. D , DNA methylation level of CpG sites related to X chromosome covered in the 450K platform; **P

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, DNA Methylation Assay, Methylation

    DNA methylation profile of CpGs (cytosine-phosphate-guanine) related to imprinted genes. A , The graph shows the DNA methylation level of CpG sites related to imprinted genes covered in the 450K platform, arranged per chromosome (β values average ranging from 0 to 1, unmethylated and fully methylated, respectively). Chromosomes 2, 4, and 8 presented methylation levels after 5-aza-CdR treatment different from DNMTs disruption (DKO cells; P
    Figure Legend Snippet: DNA methylation profile of CpGs (cytosine-phosphate-guanine) related to imprinted genes. A , The graph shows the DNA methylation level of CpG sites related to imprinted genes covered in the 450K platform, arranged per chromosome (β values average ranging from 0 to 1, unmethylated and fully methylated, respectively). Chromosomes 2, 4, and 8 presented methylation levels after 5-aza-CdR treatment different from DNMTs disruption (DKO cells; P

    Techniques Used: DNA Methylation Assay, Methylation

    Global DNA methylation analysis. A , One percent agarose gel staining with ethidium bromide showing non-digested DNA (ND) and DNA digested with Msp I or Hpa II, which is an isoschizomer of Msp I methylation sensitive enzyme, at different media concentrations of 5-aza-2′-deoxycytidine (5-aza-CdR; 0, 0.5, 1.0, and 10 µM). B , Percentage of DNA methylation of each 5-aza-CdR treatment condition and DKO cells in relation to basal methylation of the HCT116 cell line (data from 2 different assays).
    Figure Legend Snippet: Global DNA methylation analysis. A , One percent agarose gel staining with ethidium bromide showing non-digested DNA (ND) and DNA digested with Msp I or Hpa II, which is an isoschizomer of Msp I methylation sensitive enzyme, at different media concentrations of 5-aza-2′-deoxycytidine (5-aza-CdR; 0, 0.5, 1.0, and 10 µM). B , Percentage of DNA methylation of each 5-aza-CdR treatment condition and DKO cells in relation to basal methylation of the HCT116 cell line (data from 2 different assays).

    Techniques Used: DNA Methylation Assay, Agarose Gel Electrophoresis, Staining, Methylation

    8) Product Images from "Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling"

    Article Title: Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling

    Journal: Genome Biology

    doi: 10.1186/s13059-016-1066-1

    Infinium methylation probe design. a The difference in DNA methylation measurement process used by Illumina Infinium Type I and II probes is demonstrated with two probes targeting adjacent CpG sites in the BRCA1 promoter. Both probes are present on EPIC and HM450 platforms. b Infinium I (cg21253966) and Infinium II (cg04110421) probes targeting two adjacent CpG sites in the BRCA1 promoter region; the targeted CpG sites are highlighted in green . Each probe is designed to hybridise a 50 bp DNA sequence, underlined in blue, downstream of the targeted CpG site. c DNA methylation measurement with Infinium I probes is carried out by two beads – the unmethylated (U) bead measures the unmethylated signal and methylated (M) bead measures the methylated signal. The unmethylated signal detection for the cg21253966 probe is schematically represented on the left panel . Briefly, the unmethylated bead probe (U) sequence is designed to match bisulphite converted DNA sequence of the unmethylated locus. (Note that cytosines in both the target CpG site and all other CpG sites bound by the 50 bp probe are assumed to be unmethylated and therefore converted to Ts during bisulphite reaction.) The hybridisation of a bisulphite converted unmethylated DNA fragment to the bead enables single base extension and incorporation of a ddNTP labelled nucleotide matching the nucleotide immediately upstream of the target CpG site; in this case incorporation of an A nucleotide and signal detection in the RED channel . Hybridisation of the methylated bead probe (M), on the other hand, results in mismatch at the 3′ end of the probe and inhibition of single base extension. Detection of the methylated signal, shown on the right panel , follows similar steps. d For Infinium II probes the unmethylated and methylated signals are measured by the same bead (U/M). The bead probe sequence is designed to match bisulphite converted DNA of both the methylated and unmethylated locus. This is achieved by making the cytosine of the target CpG site the single base extension locus and replacing cytosines of all other CpG sites within the probe sequence with degenerate R bases that hybridises to both T (representing unmethylated and converted cytosine) and C (representing methylated and protected cytosine) bases. The unmethylated signal detection for the cg04110421 probe is schematically represented on the left panel. The hybridisation of the bisulphite converted unmethylated DNA fragment enables single base extension and incorporation of ddNTP labelled A nucleotide matching the unmethylated and converted cytosine at the target CpG site and signal detection on the RED channel . The detection of the methylation signal, shown on the right panel , is the same except that in this case single base extension results in incorporation of ddNTP labelled G nucleotide matching the methylated and protected cytosine at the target CpG site and signal detection on the GREEN channel
    Figure Legend Snippet: Infinium methylation probe design. a The difference in DNA methylation measurement process used by Illumina Infinium Type I and II probes is demonstrated with two probes targeting adjacent CpG sites in the BRCA1 promoter. Both probes are present on EPIC and HM450 platforms. b Infinium I (cg21253966) and Infinium II (cg04110421) probes targeting two adjacent CpG sites in the BRCA1 promoter region; the targeted CpG sites are highlighted in green . Each probe is designed to hybridise a 50 bp DNA sequence, underlined in blue, downstream of the targeted CpG site. c DNA methylation measurement with Infinium I probes is carried out by two beads – the unmethylated (U) bead measures the unmethylated signal and methylated (M) bead measures the methylated signal. The unmethylated signal detection for the cg21253966 probe is schematically represented on the left panel . Briefly, the unmethylated bead probe (U) sequence is designed to match bisulphite converted DNA sequence of the unmethylated locus. (Note that cytosines in both the target CpG site and all other CpG sites bound by the 50 bp probe are assumed to be unmethylated and therefore converted to Ts during bisulphite reaction.) The hybridisation of a bisulphite converted unmethylated DNA fragment to the bead enables single base extension and incorporation of a ddNTP labelled nucleotide matching the nucleotide immediately upstream of the target CpG site; in this case incorporation of an A nucleotide and signal detection in the RED channel . Hybridisation of the methylated bead probe (M), on the other hand, results in mismatch at the 3′ end of the probe and inhibition of single base extension. Detection of the methylated signal, shown on the right panel , follows similar steps. d For Infinium II probes the unmethylated and methylated signals are measured by the same bead (U/M). The bead probe sequence is designed to match bisulphite converted DNA of both the methylated and unmethylated locus. This is achieved by making the cytosine of the target CpG site the single base extension locus and replacing cytosines of all other CpG sites within the probe sequence with degenerate R bases that hybridises to both T (representing unmethylated and converted cytosine) and C (representing methylated and protected cytosine) bases. The unmethylated signal detection for the cg04110421 probe is schematically represented on the left panel. The hybridisation of the bisulphite converted unmethylated DNA fragment enables single base extension and incorporation of ddNTP labelled A nucleotide matching the unmethylated and converted cytosine at the target CpG site and signal detection on the RED channel . The detection of the methylation signal, shown on the right panel , is the same except that in this case single base extension results in incorporation of ddNTP labelled G nucleotide matching the methylated and protected cytosine at the target CpG site and signal detection on the GREEN channel

    Techniques Used: Methylation, DNA Methylation Assay, Sequencing, Hybridization, Inhibition

    9) Product Images from "High-Risk Human Papillomavirus E7 Alters Host DNA Methylome and Represses HLA-E Expression in Human Keratinocytes"

    Article Title: High-Risk Human Papillomavirus E7 Alters Host DNA Methylome and Represses HLA-E Expression in Human Keratinocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03295-7

    HPV16 E7 alters host genome methylation in keratinocytes. Global DNA methylation profiles in NIKS, NIKS-16, NIKS-18, and NIKS-16ΔE7 cells were analyzed in triplicate using Illumina Infinium HumanMethylation450 BeadChip arrays. ( a ) Principal component analysis data are shown for each replicate of normalized data from NIKS (red circle), NIKS-16 (blue square), NIKS-18 (green triangle) and NIKS-16ΔE7 (black triangle) cells. ( b ) Methylation array data from NIKS (black), NIKS-16 (red), NIKS-18 (orange) and NIKS-16ΔE7 (blue) cells were normalized using SWAN and the relative methylation (β) density across the genome are plotted. β represents the ratio of methylated signal to total signal (methylated + unmethylated) at a given CpG site. β near 0 or 1 indicates no methylation or complete methylation, respectively. Three pairwise comparisons are summarized by Venn diagrams showing the number of overlapping ( c ) differentially methylated positions (DMP, FDR adjusted p
    Figure Legend Snippet: HPV16 E7 alters host genome methylation in keratinocytes. Global DNA methylation profiles in NIKS, NIKS-16, NIKS-18, and NIKS-16ΔE7 cells were analyzed in triplicate using Illumina Infinium HumanMethylation450 BeadChip arrays. ( a ) Principal component analysis data are shown for each replicate of normalized data from NIKS (red circle), NIKS-16 (blue square), NIKS-18 (green triangle) and NIKS-16ΔE7 (black triangle) cells. ( b ) Methylation array data from NIKS (black), NIKS-16 (red), NIKS-18 (orange) and NIKS-16ΔE7 (blue) cells were normalized using SWAN and the relative methylation (β) density across the genome are plotted. β represents the ratio of methylated signal to total signal (methylated + unmethylated) at a given CpG site. β near 0 or 1 indicates no methylation or complete methylation, respectively. Three pairwise comparisons are summarized by Venn diagrams showing the number of overlapping ( c ) differentially methylated positions (DMP, FDR adjusted p

    Techniques Used: Methylation, DNA Methylation Assay

    10) Product Images from "Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae)"

    Article Title: Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae)

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07231-7

    Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for Msp I/ Hpa II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.
    Figure Legend Snippet: Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for Msp I/ Hpa II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.

    Techniques Used: Sequencing, Derivative Assay, Polymerase Chain Reaction, Amplification

    PCR amplification and Southern blot hybridisation of SSUsat in different bivalve species. Agarose gel electrophoresis of the SSUsat fragments obtained after PCR amplifying genomic DNA of Spisula subtruncata (SSU), Spisula solida (SSO), Mactra stultorum (MST) and Donax trunculus (DTR) using primers derived from the SSUsat consensus sequence (a) . Corresponding Southern blot hybridisation using an SSUsat probe ( b ). Blank represents PCR reaction without DNA template. ( c ) Scheme of tandemly organized SSUsat monomers showing the locations of the inversely orientated PCR primers (SSUsatPR1 in red, SSUsatPR2 in green).
    Figure Legend Snippet: PCR amplification and Southern blot hybridisation of SSUsat in different bivalve species. Agarose gel electrophoresis of the SSUsat fragments obtained after PCR amplifying genomic DNA of Spisula subtruncata (SSU), Spisula solida (SSO), Mactra stultorum (MST) and Donax trunculus (DTR) using primers derived from the SSUsat consensus sequence (a) . Corresponding Southern blot hybridisation using an SSUsat probe ( b ). Blank represents PCR reaction without DNA template. ( c ) Scheme of tandemly organized SSUsat monomers showing the locations of the inversely orientated PCR primers (SSUsatPR1 in red, SSUsatPR2 in green).

    Techniques Used: Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Microscale Thermophoresis, Derivative Assay, Sequencing

    Southern blot hybridisation analysis of SSUsat repeats in Spisula subtruncata . Agarose gel electrophoresis of Pvu II and Hae III digested genomic DNA of Spisula subtruncata showing ladder-like multimer bands of a 315 bp monomer unit ( a ). After being Southern blotted on a nitrocellulose membrane, the electrophoresed DNA was hybridised with an SSUsat monomer probe yielding identical ladder-like multimer bands of a 315 bp monomer unit ( b ).
    Figure Legend Snippet: Southern blot hybridisation analysis of SSUsat repeats in Spisula subtruncata . Agarose gel electrophoresis of Pvu II and Hae III digested genomic DNA of Spisula subtruncata showing ladder-like multimer bands of a 315 bp monomer unit ( a ). After being Southern blotted on a nitrocellulose membrane, the electrophoresed DNA was hybridised with an SSUsat monomer probe yielding identical ladder-like multimer bands of a 315 bp monomer unit ( b ).

    Techniques Used: Southern Blot, Hybridization, Agarose Gel Electrophoresis

    11) Product Images from "Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation"

    Article Title: Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx601

    The bidirectional Tet promoter is methylated in the ROSA26 locus and can be reactivated by DNMTi ( A ) GFP expression in BidiTet-Luc/GFP ES cells upon cultivation in presence of the DNMTis Decitabine and Azacytidine. BidiTet-Luc/GFP ES cells were cultivated in presence or absence of Doxycycline and Decitabine or Azacytidine for three days and subsequently subjected to flow cytometry. The percentage of GFP expressing cells is depicted. ( B ) In vivo imaging of induced BidiTet-Luc/GFP mice upon treatment with Azacytidine. Whole body bioluminescence was determined in three independent animals before and three days after the first treatment with Azacytidine. As control, Rosa-Luc animals were used. The bioluminescence of three independent mice per group is depicted on the left. On day 3, the liver was isolated and slices were stained for GFP expression. Depicted are representative sections from mice of the indicated groups. ( C ) Schematic depiction of the bidirectional Tet promoter in BidiTet-Luc/GFP cells and the position of the CpG's. The bidirectional Tet promoter comprises 30 CpGs, 8 CpG's flank the tetO sequences, 3 CpGs are located in a spacer element and the remaining 19 CpG are in the two opposing CMV promoter sequences. The location of the CpGs within the promoter (lines) and within the amplified PCR fragment (lollipops) are indicated. Note that the 5′ minimal CMV promoter is slightly shorter than the 3′ CMV promoter ( 72 ). ( D ) Methylation status of the Tet promoter in BidiTet-Luc/GFP ES cells. Cells were treated for 3 days with Decitabine. GFP positive and negative cells were sorted, DNA was extracted and subjected to bisulfite analysis. Subsequently, the Tet promoter DNA was PCR amplified and cloned in E. coli. Sequences of eight randomly picked clones representing eight independent cells are depicted. In each line, the circles depict the 17 CpGs of an individual clone/cell. Black circles indicate methylated cytosines and non-filled circles non-methylated cytosines. ( E ) Methylation status of Tet promoter in BidiTet-Luc/GFP mice. Bisulfite analysis of the Tet promoter based on DNA extracted from mouse livers. The methylation status of the Tet promoter in the non-induced (–Dox), induced (+Dox) and Azacytidine/Dox induced animals (+Dox +Aza) is depicted. Three independent mice per group were analysed. For each mouse, eight independent cells were sequenced and analysed. Black circles indicate methylated cytosines and non-filled circles non methylated cytosines. ( F ) Epityper based DNA methylation analysis of the ROSA26 and ThumpD3 promoter in cells and mice. The heat maps represent the results of the methylation analysis done on Epityper. The various CpG motifs of the two promoters are indicated on the x-axis of each map and the samples used are indicated on the y-axis. The reference scale is indicated (0.01 = 1% methylation and 0.26 = 26% methylation). Each vertical column represents the same CpG motif analyzed from different gDNA samples while each row represents the different CpGs within the same sample.
    Figure Legend Snippet: The bidirectional Tet promoter is methylated in the ROSA26 locus and can be reactivated by DNMTi ( A ) GFP expression in BidiTet-Luc/GFP ES cells upon cultivation in presence of the DNMTis Decitabine and Azacytidine. BidiTet-Luc/GFP ES cells were cultivated in presence or absence of Doxycycline and Decitabine or Azacytidine for three days and subsequently subjected to flow cytometry. The percentage of GFP expressing cells is depicted. ( B ) In vivo imaging of induced BidiTet-Luc/GFP mice upon treatment with Azacytidine. Whole body bioluminescence was determined in three independent animals before and three days after the first treatment with Azacytidine. As control, Rosa-Luc animals were used. The bioluminescence of three independent mice per group is depicted on the left. On day 3, the liver was isolated and slices were stained for GFP expression. Depicted are representative sections from mice of the indicated groups. ( C ) Schematic depiction of the bidirectional Tet promoter in BidiTet-Luc/GFP cells and the position of the CpG's. The bidirectional Tet promoter comprises 30 CpGs, 8 CpG's flank the tetO sequences, 3 CpGs are located in a spacer element and the remaining 19 CpG are in the two opposing CMV promoter sequences. The location of the CpGs within the promoter (lines) and within the amplified PCR fragment (lollipops) are indicated. Note that the 5′ minimal CMV promoter is slightly shorter than the 3′ CMV promoter ( 72 ). ( D ) Methylation status of the Tet promoter in BidiTet-Luc/GFP ES cells. Cells were treated for 3 days with Decitabine. GFP positive and negative cells were sorted, DNA was extracted and subjected to bisulfite analysis. Subsequently, the Tet promoter DNA was PCR amplified and cloned in E. coli. Sequences of eight randomly picked clones representing eight independent cells are depicted. In each line, the circles depict the 17 CpGs of an individual clone/cell. Black circles indicate methylated cytosines and non-filled circles non-methylated cytosines. ( E ) Methylation status of Tet promoter in BidiTet-Luc/GFP mice. Bisulfite analysis of the Tet promoter based on DNA extracted from mouse livers. The methylation status of the Tet promoter in the non-induced (–Dox), induced (+Dox) and Azacytidine/Dox induced animals (+Dox +Aza) is depicted. Three independent mice per group were analysed. For each mouse, eight independent cells were sequenced and analysed. Black circles indicate methylated cytosines and non-filled circles non methylated cytosines. ( F ) Epityper based DNA methylation analysis of the ROSA26 and ThumpD3 promoter in cells and mice. The heat maps represent the results of the methylation analysis done on Epityper. The various CpG motifs of the two promoters are indicated on the x-axis of each map and the samples used are indicated on the y-axis. The reference scale is indicated (0.01 = 1% methylation and 0.26 = 26% methylation). Each vertical column represents the same CpG motif analyzed from different gDNA samples while each row represents the different CpGs within the same sample.

    Techniques Used: Methylation, Expressing, Flow Cytometry, Cytometry, In Vivo Imaging, Mouse Assay, Isolation, Staining, Amplification, Polymerase Chain Reaction, Clone Assay, DNA Methylation Assay

    12) Product Images from "Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion"

    Article Title: Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion

    Journal: Genetics & Epigenetics

    doi: 10.1177/1179237X17736107

    Comparison and classification of the DNA methylation patterns. (A) The DNA methylation pattern in buffy coats (red line) and CVS (blue line), represented by mXXXXXXXXXX; 0 or 1 is the unmethylated or methylated status of a CpG site, respectively. The consensus patterns were used to classify the data into 3 groups: the group of relative complement patterns of CVS in buffy coat (A area), the group of intersecting patterns of CVS and buffy coat (B area), and the group of relative complementary patterns of buffy coat in CVS (C area). (B) The Venn diagram shows the intersecting part of the 2 different types of sample in the green area. CVS indicates chorionic villus samples.
    Figure Legend Snippet: Comparison and classification of the DNA methylation patterns. (A) The DNA methylation pattern in buffy coats (red line) and CVS (blue line), represented by mXXXXXXXXXX; 0 or 1 is the unmethylated or methylated status of a CpG site, respectively. The consensus patterns were used to classify the data into 3 groups: the group of relative complement patterns of CVS in buffy coat (A area), the group of intersecting patterns of CVS and buffy coat (B area), and the group of relative complementary patterns of buffy coat in CVS (C area). (B) The Venn diagram shows the intersecting part of the 2 different types of sample in the green area. CVS indicates chorionic villus samples.

    Techniques Used: DNA Methylation Assay, Methylation

    DNA methylation profile of individual CpG sites. (A) Heat map for DNA methylation levels on individual CpG sites. The dendrogram shows clustering data by similarity of profile into 2 groups: buffy coats (red line) and the CVS group (green line). The labels of the dendrogram branches give sample details: S and number are sample codes, F or M are sexes, and wks refers to the gestation age in weeks. (B) A box plot of the methylation levels of individual CpG sites. The green boxes are buffy coat samples, and the red boxes are CVS. CVS indicates chorionic villus samples.
    Figure Legend Snippet: DNA methylation profile of individual CpG sites. (A) Heat map for DNA methylation levels on individual CpG sites. The dendrogram shows clustering data by similarity of profile into 2 groups: buffy coats (red line) and the CVS group (green line). The labels of the dendrogram branches give sample details: S and number are sample codes, F or M are sexes, and wks refers to the gestation age in weeks. (B) A box plot of the methylation levels of individual CpG sites. The green boxes are buffy coat samples, and the red boxes are CVS. CVS indicates chorionic villus samples.

    Techniques Used: DNA Methylation Assay, Methylation

    Comparison of DNA methylation profiles of the SEA breakpoint junction. (A) A total of 53 DNA samples from buffy coat and 10 CVS were amplified across the breakpoint region, and then high-resolution melting analysis was performed; the green and red lines show the HRM pattern of buffy coat and CVS, respectively. The yellow line served as a nonmethylated DNA control. (B) The box plot shows the different DNA methylation levels between the 2 types of sample. Statistical analysis comparing sample groups was conducted using a 1-tailed Student t test with unequal variance. The mean was significantly different ( P
    Figure Legend Snippet: Comparison of DNA methylation profiles of the SEA breakpoint junction. (A) A total of 53 DNA samples from buffy coat and 10 CVS were amplified across the breakpoint region, and then high-resolution melting analysis was performed; the green and red lines show the HRM pattern of buffy coat and CVS, respectively. The yellow line served as a nonmethylated DNA control. (B) The box plot shows the different DNA methylation levels between the 2 types of sample. Statistical analysis comparing sample groups was conducted using a 1-tailed Student t test with unequal variance. The mean was significantly different ( P

    Techniques Used: DNA Methylation Assay, Amplification

    13) Product Images from "DNA methylation alterations in iPSC- and hESC-derived neurons: potential implications for neurological disease modeling"

    Article Title: DNA methylation alterations in iPSC- and hESC-derived neurons: potential implications for neurological disease modeling

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-018-0440-0

    Genome-wide and single gene DNA methylation analysis. a Volcano plot showing CpG methylation changes during neuronal differentiation. HESC- and iPSC-derived NSC and neurons were grouped together due to their high similarity, respectively. We calculated the difference for each NSC and neuronal pair (Δ) based on a paired t test of p
    Figure Legend Snippet: Genome-wide and single gene DNA methylation analysis. a Volcano plot showing CpG methylation changes during neuronal differentiation. HESC- and iPSC-derived NSC and neurons were grouped together due to their high similarity, respectively. We calculated the difference for each NSC and neuronal pair (Δ) based on a paired t test of p

    Techniques Used: Genome Wide, DNA Methylation Assay, CpG Methylation Assay, Derivative Assay

    Single gene DNA methylation analysis. a - e DNA methylation levels (%) of individual CpGs of single genes from isogenic hESC-derived and iPSC-derived NSC and neurons ( APP , GNAS , MIR886 , PMP22 , SNCA intron 1). The % difference at each CpG between hESC-derived and iPSC-derived neurons is depicted at the secondary x-axis. Neurons were differentiated for 6 weeks. Blue = hESC-derived NSC or neurons; red = iPSC-derived NSC or neurons
    Figure Legend Snippet: Single gene DNA methylation analysis. a - e DNA methylation levels (%) of individual CpGs of single genes from isogenic hESC-derived and iPSC-derived NSC and neurons ( APP , GNAS , MIR886 , PMP22 , SNCA intron 1). The % difference at each CpG between hESC-derived and iPSC-derived neurons is depicted at the secondary x-axis. Neurons were differentiated for 6 weeks. Blue = hESC-derived NSC or neurons; red = iPSC-derived NSC or neurons

    Techniques Used: DNA Methylation Assay, Derivative Assay

    Genome-wide DNA methylation analysis. a Pairwise correlation plots (Pearson correlation, genome-wide DNA methylation analysis) of hESC- and iPSC-derived NSC and neurons display high correlation coefficients (black: hES-NSC vs. iPS-NSC, blue: hES-Neurons vs. iPS-Neurons) and minimal interclonal variance (orange: comparison of iPS-NSC clone 1, clone 2, and clone 3; red: comparison of iPS-Neurons clone 1, clone 2, and clone 3). b Number of differentially hypo- and hypermethylated CpGs (DMCG) in iPS-Neurons compared to hES-Neurons. iPSC-derived neurons demonstrate slightly more hyper- than hypomethylated DMCG compared to hESC-derived neurons. c Annotation of hypo- and hypermethylated DMCG in terms of gene regulatory regions (intergenic gene regions, 1st exon, 3′ and 5′ untranslated region (UTR), gene body, promoter areas: transcription start sites (TSS) 1500 and 200; upper pie charts), and CpG islands (CpG islands and flanking regions before (N_Shelf, N_Shore) and after (S_Shelf, S_Shore) CpG islands; lower pie charts) in comparison to the overall distribution of markers on the whole 450 K array (left pie charts), respectively. d , e Interclonal variation of DNA methylation is pronounced at CpGs with intermediate methylation levels in iPS-Neurons ( d ) and -NSC ( e ), respectively. f Variation of DMCG is highly correlated between iPS-NSC and iPS-Neurons. SD, standard deviation
    Figure Legend Snippet: Genome-wide DNA methylation analysis. a Pairwise correlation plots (Pearson correlation, genome-wide DNA methylation analysis) of hESC- and iPSC-derived NSC and neurons display high correlation coefficients (black: hES-NSC vs. iPS-NSC, blue: hES-Neurons vs. iPS-Neurons) and minimal interclonal variance (orange: comparison of iPS-NSC clone 1, clone 2, and clone 3; red: comparison of iPS-Neurons clone 1, clone 2, and clone 3). b Number of differentially hypo- and hypermethylated CpGs (DMCG) in iPS-Neurons compared to hES-Neurons. iPSC-derived neurons demonstrate slightly more hyper- than hypomethylated DMCG compared to hESC-derived neurons. c Annotation of hypo- and hypermethylated DMCG in terms of gene regulatory regions (intergenic gene regions, 1st exon, 3′ and 5′ untranslated region (UTR), gene body, promoter areas: transcription start sites (TSS) 1500 and 200; upper pie charts), and CpG islands (CpG islands and flanking regions before (N_Shelf, N_Shore) and after (S_Shelf, S_Shore) CpG islands; lower pie charts) in comparison to the overall distribution of markers on the whole 450 K array (left pie charts), respectively. d , e Interclonal variation of DNA methylation is pronounced at CpGs with intermediate methylation levels in iPS-Neurons ( d ) and -NSC ( e ), respectively. f Variation of DMCG is highly correlated between iPS-NSC and iPS-Neurons. SD, standard deviation

    Techniques Used: Genome Wide, DNA Methylation Assay, Derivative Assay, Methylation, Standard Deviation

    14) Product Images from "DNA methylation of hepatic iron sensing genes and the regulation of hepcidin expression"

    Article Title: DNA methylation of hepatic iron sensing genes and the regulation of hepcidin expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197863

    Methylation of TFR2 alpha promoter in human cell lines. A 246-bp fragment of the human TFR2 alpha promoter was generated by PCR. Amplicons contained a ClaI restriction digest site which will cut the amplicon into 138 108 bp fragments if the CpG within the digest site is methylated. Agarose gel ( a ) shows representative bands from uncut (U; water replacing enzyme) and ClaI-digested bisulphite-converted DNA from K562, HepG2, Huh7 and Jurkat cells. The arrow indicates the position of the full length amplicon (246 bp); * indicates the position of the ClaI digested fragments (138 108 bp). The cartoon depicts a representation of TFR2 alpha gene organization and the 246 bp PCR amplicon ( b ). The 5’ upstream flanking region (from -152 bp relative to the translation start site) and intron 1 (downstream to +92 bp relative to the translation site) are shown as lines; exon 1 containing the promoter and the translated region are shown as boxes. The vertical arrow denotes the ClaI digest site. Horizontal lines below represent bisulphite sequencing of individual amplicons in each cell line. Open circles indicate unmethylated CpGs, filled circles represent methylated CpGs.
    Figure Legend Snippet: Methylation of TFR2 alpha promoter in human cell lines. A 246-bp fragment of the human TFR2 alpha promoter was generated by PCR. Amplicons contained a ClaI restriction digest site which will cut the amplicon into 138 108 bp fragments if the CpG within the digest site is methylated. Agarose gel ( a ) shows representative bands from uncut (U; water replacing enzyme) and ClaI-digested bisulphite-converted DNA from K562, HepG2, Huh7 and Jurkat cells. The arrow indicates the position of the full length amplicon (246 bp); * indicates the position of the ClaI digested fragments (138 108 bp). The cartoon depicts a representation of TFR2 alpha gene organization and the 246 bp PCR amplicon ( b ). The 5’ upstream flanking region (from -152 bp relative to the translation start site) and intron 1 (downstream to +92 bp relative to the translation site) are shown as lines; exon 1 containing the promoter and the translated region are shown as boxes. The vertical arrow denotes the ClaI digest site. Horizontal lines below represent bisulphite sequencing of individual amplicons in each cell line. Open circles indicate unmethylated CpGs, filled circles represent methylated CpGs.

    Techniques Used: Methylation, Generated, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Bisulfite Sequencing

    Methylation of HAMP promoter. Bisulphite-converted DNA from HepG2 and Huh7 was subjected to qRT-PCR using a combination of primers specific for methylated and unmethylated sequences. Data are presented as relative levels; ΔCt relative to the methylated forward: methylated reverse primer set (M-M) for HepG2 cells.
    Figure Legend Snippet: Methylation of HAMP promoter. Bisulphite-converted DNA from HepG2 and Huh7 was subjected to qRT-PCR using a combination of primers specific for methylated and unmethylated sequences. Data are presented as relative levels; ΔCt relative to the methylated forward: methylated reverse primer set (M-M) for HepG2 cells.

    Techniques Used: Methylation, Quantitative RT-PCR

    15) Product Images from "Circular RNA CpG island hypermethylation-associated silencing in human cancer"

    Article Title: Circular RNA CpG island hypermethylation-associated silencing in human cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25673

    Screening for CpG island hypermethylation-associated silencing of circular RNAs in cancer cells ( A ) Flow-chart used to identify candidate circRNAs silenced in colon cancer through CpG island hypermethylation in the promoter region of their host genes. FC, Fold Change; SNP, Single Nucleotide Polymorphism. ( B ) DNA methylation profile of the 5′-end CpG island regulatory region for the TUSC3, ATRNL1, POMT1 or SAMD4A genes analyzed by the 450K DNA methylation microarray. DNA methylation data for healthy colon mucosa correspond to two normal colon patient samples available at TCGA (Normal colon 1: TCGA-A6-2675-11A, sigmoid colon normal tissue; Normal colon 2: TCGA-A6-2685-11A, sigmoid colon normal tissue). Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from normal colon, HCT-116 and DKO cells are shown.
    Figure Legend Snippet: Screening for CpG island hypermethylation-associated silencing of circular RNAs in cancer cells ( A ) Flow-chart used to identify candidate circRNAs silenced in colon cancer through CpG island hypermethylation in the promoter region of their host genes. FC, Fold Change; SNP, Single Nucleotide Polymorphism. ( B ) DNA methylation profile of the 5′-end CpG island regulatory region for the TUSC3, ATRNL1, POMT1 or SAMD4A genes analyzed by the 450K DNA methylation microarray. DNA methylation data for healthy colon mucosa correspond to two normal colon patient samples available at TCGA (Normal colon 1: TCGA-A6-2675-11A, sigmoid colon normal tissue; Normal colon 2: TCGA-A6-2685-11A, sigmoid colon normal tissue). Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from normal colon, HCT-116 and DKO cells are shown.

    Techniques Used: Flow Cytometry, DNA Methylation Assay, Microarray, Methylation

    16) Product Images from "DNA methylation-dependent repression of PDZ-LIM domain-containing protein 2 in colon cancer and its role as a potential therapeutic target"

    Article Title: DNA methylation-dependent repression of PDZ-LIM domain-containing protein 2 in colon cancer and its role as a potential therapeutic target

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-09-3263

    PDLIM2 repression in colon cancer cells involves DNA methylation. A , RNA levels of DNMT1, DNMT3a and DNMT3b in the indicated colon cancer cells were analyzed by real-time PCR using β-actin mRNA level as a control and represented as fold induction in mRNA abundance relative to those in MCF10A cells (set as 1). The data presented are the mean ± standard deviation (n = 3). B , The indicated cell lines were treated with the DNMT inhibitor 5-aza-dC (5μM) for 48 h, followed by real-time PCR to determine relative mRNA levels of PDLIM2. Changes in PDLIM2 mRNA abundance following 5-aza-dC treatment are represented as fold induction relative to those observed in an RNA sample from mock-treated cells. C , The indicated colon cancer cell lines were treated with 5 μM 5-aza-dC or vehicle for the indicated time points, followed by cell growth assay. D , The indicated cell lines were treated with 5 μM 5-aza-dC or vehicle for 5 days, followed by the bisulfite genomic DNA sequencing as described in Material and Methods. Each circle represents a CpG site; open circles represent unmethylated CpG dimucleotides whereas filled circles represent methylated CpG sites. The ratios of the filled area in circles represent percentiles of the methylation in the CpG sites. The position of each CpG nucleotide relative to the PDLIM2 transcription initiation site (+1) is indicated at the top.
    Figure Legend Snippet: PDLIM2 repression in colon cancer cells involves DNA methylation. A , RNA levels of DNMT1, DNMT3a and DNMT3b in the indicated colon cancer cells were analyzed by real-time PCR using β-actin mRNA level as a control and represented as fold induction in mRNA abundance relative to those in MCF10A cells (set as 1). The data presented are the mean ± standard deviation (n = 3). B , The indicated cell lines were treated with the DNMT inhibitor 5-aza-dC (5μM) for 48 h, followed by real-time PCR to determine relative mRNA levels of PDLIM2. Changes in PDLIM2 mRNA abundance following 5-aza-dC treatment are represented as fold induction relative to those observed in an RNA sample from mock-treated cells. C , The indicated colon cancer cell lines were treated with 5 μM 5-aza-dC or vehicle for the indicated time points, followed by cell growth assay. D , The indicated cell lines were treated with 5 μM 5-aza-dC or vehicle for 5 days, followed by the bisulfite genomic DNA sequencing as described in Material and Methods. Each circle represents a CpG site; open circles represent unmethylated CpG dimucleotides whereas filled circles represent methylated CpG sites. The ratios of the filled area in circles represent percentiles of the methylation in the CpG sites. The position of each CpG nucleotide relative to the PDLIM2 transcription initiation site (+1) is indicated at the top.

    Techniques Used: DNA Methylation Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Growth Assay, DNA Sequencing, Methylation

    17) Product Images from "Genome Wide Peripheral Blood Leukocyte DNA Methylation Microarrays Identified a Single Association with Inflammatory Bowel Diseases"

    Article Title: Genome Wide Peripheral Blood Leukocyte DNA Methylation Microarrays Identified a Single Association with Inflammatory Bowel Diseases

    Journal: Inflammatory bowel diseases

    doi: 10.1002/ibd.22956

    PBL DNA Samples
    Figure Legend Snippet: PBL DNA Samples

    Techniques Used:

    18) Product Images from "Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors"

    Article Title: Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6140

    DNA hypermethylation in areas with GP4 cribriform architecture and/or IDC
    Figure Legend Snippet: DNA hypermethylation in areas with GP4 cribriform architecture and/or IDC

    Techniques Used:

    19) Product Images from "High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation"

    Article Title: High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00723-15

    Analysis of chromatin accessibility in the murine β-globin gene locus using MAPit. (A) Diagram of the murine β-globin gene locus. The murine β-globin locus consists of an LCR harboring multiple DNase I HSs (HS1 to HS6) and developmentally regulated globin genes (Ey-, bh1-, and bh2-globin genes, as well as adult βmaj- and βmin-globin genes). (B) Fraction of benzidine-positive MEL cells at different time points after induction of differentiation. The error bar reflects the average of the number of benzidine-positive cells counted in four different windows from two independent experiments. (C) Diagram of the MAPit procedure. Nuclei are isolated and incubated with the GpC-specific DNMT M.CviPI. Genomic DNA is extracted, bisulfite converted, amplified using bar-coded primers, and subjected to high-throughput sequencing. Using specific software, individual sequencing reads are analyzed and subjected to unsupervised hierarchical clustering to visualize the pattern of methylated and unmethylated GCs present in each probed chromatin sample. Yellow indicates the accessible spans of chromatin in which two or more subsequent GCs are methylated. Black indicates either a footprint in which two or more subsequent GCs over a known transcription factor binding site are unmethylated and flanked on both sides by at least one methylated GC or inaccessible chromatin in which multiple GCs are unmethylated. Gray represents the border between a methylated and an unmethylated GC. (D) MAPit analysis of the Mlh1 gene promoter. MEL cells were treated with DMSO to induce differentiation and were subjected to MAPit at various time points, as indicated. DNA was bisulfite converted and amplified by Mlh1 -specific primers. The amplicons were subcloned and sequenced. The arrows indicate the TSS, and the bar at the bottom indicates 147 bp, the length of DNA within a nucleosome core particle. (E) MAPit analysis of the embryonic Ey-globin genes. MEL cells were induced to differentiate and were subjected to MAPit and high-throughput sequencing at the indicated time points. The arrows indicate the TSS.
    Figure Legend Snippet: Analysis of chromatin accessibility in the murine β-globin gene locus using MAPit. (A) Diagram of the murine β-globin gene locus. The murine β-globin locus consists of an LCR harboring multiple DNase I HSs (HS1 to HS6) and developmentally regulated globin genes (Ey-, bh1-, and bh2-globin genes, as well as adult βmaj- and βmin-globin genes). (B) Fraction of benzidine-positive MEL cells at different time points after induction of differentiation. The error bar reflects the average of the number of benzidine-positive cells counted in four different windows from two independent experiments. (C) Diagram of the MAPit procedure. Nuclei are isolated and incubated with the GpC-specific DNMT M.CviPI. Genomic DNA is extracted, bisulfite converted, amplified using bar-coded primers, and subjected to high-throughput sequencing. Using specific software, individual sequencing reads are analyzed and subjected to unsupervised hierarchical clustering to visualize the pattern of methylated and unmethylated GCs present in each probed chromatin sample. Yellow indicates the accessible spans of chromatin in which two or more subsequent GCs are methylated. Black indicates either a footprint in which two or more subsequent GCs over a known transcription factor binding site are unmethylated and flanked on both sides by at least one methylated GC or inaccessible chromatin in which multiple GCs are unmethylated. Gray represents the border between a methylated and an unmethylated GC. (D) MAPit analysis of the Mlh1 gene promoter. MEL cells were treated with DMSO to induce differentiation and were subjected to MAPit at various time points, as indicated. DNA was bisulfite converted and amplified by Mlh1 -specific primers. The amplicons were subcloned and sequenced. The arrows indicate the TSS, and the bar at the bottom indicates 147 bp, the length of DNA within a nucleosome core particle. (E) MAPit analysis of the embryonic Ey-globin genes. MEL cells were induced to differentiate and were subjected to MAPit and high-throughput sequencing at the indicated time points. The arrows indicate the TSS.

    Techniques Used: Isolation, Incubation, Gel Permeation Chromatography, Amplification, Next-Generation Sequencing, Software, Sequencing, Methylation, Binding Assay

    20) Product Images from "Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase"

    Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007282

    PVX-based expression of C4 reverses TGS of a GFP transgene and suppresses endogenous and exogenous DNA methylation. (A) 16-TGS plants were inoculated with PVX-C4-HA, PVX-C4 R13A -HA or PVX-cLUC-HA, and photographed under UV light at 14 dpi. cLUC represents c-terminal fragment of the firefly luciferase. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as a protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Values represent means ± SE from three independent experiments. (*p
    Figure Legend Snippet: PVX-based expression of C4 reverses TGS of a GFP transgene and suppresses endogenous and exogenous DNA methylation. (A) 16-TGS plants were inoculated with PVX-C4-HA, PVX-C4 R13A -HA or PVX-cLUC-HA, and photographed under UV light at 14 dpi. cLUC represents c-terminal fragment of the firefly luciferase. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as a protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Values represent means ± SE from three independent experiments. (*p

    Techniques Used: Expressing, DNA Methylation Assay, Luciferase, Western Blot, Staining, Quantitative RT-PCR

    Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p
    Figure Legend Snippet: Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

    Techniques Used: Infection, Plasmid Preparation, Southern Blot, Labeling, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

    Silencing of NbSAMS2 reverses TGS and PTGS of GFP. (A) 16-TGS plants were co-inoculated with TYLCCNV and βM2 vector containing DNA fragment of NbSAMS2 or LUC. Plants were photographed under UV light at 21 dpi. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as an equal protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Data were obtained from three independent experiments. Values represent means ± SE from three independent experiments. (*p
    Figure Legend Snippet: Silencing of NbSAMS2 reverses TGS and PTGS of GFP. (A) 16-TGS plants were co-inoculated with TYLCCNV and βM2 vector containing DNA fragment of NbSAMS2 or LUC. Plants were photographed under UV light at 21 dpi. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as an equal protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Data were obtained from three independent experiments. Values represent means ± SE from three independent experiments. (*p

    Techniques Used: Plasmid Preparation, Western Blot, Staining, Quantitative RT-PCR

    A R13A point mutation in C4 attenuates CLCuMuV infection. (A) Schematic representation of the CLCuMuV C4 and Rep ORF. Sequences in light blue color represents mutation while red color for original nucleotide and relevant amino acids was displayed with green color. (B) Relative viral accumulation of CLCuMuV DNA. Protoplasts from the leaves of N . benthamiana plants were isolated, and then transfected with CLCuMuVΔRep and expression construct of either Rep or RepE65G. Real-time PCR analysis of V1 gene from CLCuMuVΔRep was used to determine viral DNA level. eIF4α was used as an internal control. Values represent means ± SE from three independent experiments. (C) The mutant CLCuMuV carrying C4 R13A (CLCuMuV-C4 R13A ), which contains a R13A mutation in C4 , showed the decreased viral symptom compared to wild type CLCuMuV. Photographs were taken at 21 dpi. (D) Relative viral accumulation of CLCuMuV DNA. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p
    Figure Legend Snippet: A R13A point mutation in C4 attenuates CLCuMuV infection. (A) Schematic representation of the CLCuMuV C4 and Rep ORF. Sequences in light blue color represents mutation while red color for original nucleotide and relevant amino acids was displayed with green color. (B) Relative viral accumulation of CLCuMuV DNA. Protoplasts from the leaves of N . benthamiana plants were isolated, and then transfected with CLCuMuVΔRep and expression construct of either Rep or RepE65G. Real-time PCR analysis of V1 gene from CLCuMuVΔRep was used to determine viral DNA level. eIF4α was used as an internal control. Values represent means ± SE from three independent experiments. (C) The mutant CLCuMuV carrying C4 R13A (CLCuMuV-C4 R13A ), which contains a R13A mutation in C4 , showed the decreased viral symptom compared to wild type CLCuMuV. Photographs were taken at 21 dpi. (D) Relative viral accumulation of CLCuMuV DNA. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

    Techniques Used: Mutagenesis, Infection, Isolation, Transfection, Expressing, Construct, Real-time Polymerase Chain Reaction

    21) Product Images from "Epigenetic deregulation of GATA3 in neuroblastoma is associated with increased GATA3 protein expression and with poor outcomes"

    Article Title: Epigenetic deregulation of GATA3 in neuroblastoma is associated with increased GATA3 protein expression and with poor outcomes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55382-6

    GATA3 DNA methylation and expression. ( A ) GATA3 sense (hatched bars) and antisense (unfilled bars) RNA expression assayed by QPCR, and DNA methylation levels (black bars) detected by pyrosequencing, in control tissues and neuroblastoma cell lines. RNA levels were normalized to endogenous levels of TBP and expressed relative to hNCC. DNA methylation was calculated as the average of the 01 and 02 pyrosequencing assays. ( B ) GATA3 protein levels assayed by Western blot in normal tissues (NT) and type-S, type-I, I + S and type-N neuroblastoma cell lines, with ACTIN as a loading control. Uncropped blots are shown in Supplementary Fig. S10 . ( C ) GATA3 sense RNA expression in DRG/SG cells treated with 2 μM AZA for 6 days. RNA levels were normalized to endogenous levels of TBP and expressed relative to control. Mean ± S.E.M of three experiments; *p
    Figure Legend Snippet: GATA3 DNA methylation and expression. ( A ) GATA3 sense (hatched bars) and antisense (unfilled bars) RNA expression assayed by QPCR, and DNA methylation levels (black bars) detected by pyrosequencing, in control tissues and neuroblastoma cell lines. RNA levels were normalized to endogenous levels of TBP and expressed relative to hNCC. DNA methylation was calculated as the average of the 01 and 02 pyrosequencing assays. ( B ) GATA3 protein levels assayed by Western blot in normal tissues (NT) and type-S, type-I, I + S and type-N neuroblastoma cell lines, with ACTIN as a loading control. Uncropped blots are shown in Supplementary Fig. S10 . ( C ) GATA3 sense RNA expression in DRG/SG cells treated with 2 μM AZA for 6 days. RNA levels were normalized to endogenous levels of TBP and expressed relative to control. Mean ± S.E.M of three experiments; *p

    Techniques Used: DNA Methylation Assay, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Western Blot

    GATA3 DNA methylation in neuroblastoma. ( A ) GATA3 DNA methylation detected by MCIP. Black bars show the probe ratios derived from MCIP for hNCC and four neuroblastoma cell lines, positioned on the GATA3 CpG island promoter region, showing the sense and antisense transcripts and CpG island (CGI) (human genome build NCBI36/Hg18 visualised on the UCSC genome browser; http://genome.ucsc.edu ). The positions of the hypomethylated region and the two pyrosequencing assays (01 and 02) are shown in red at the top. ( B ) Dotboxplot of GATA3 antisense DNA methylation measured by pyrosequencing in normal tissues (NT, n = 4), neuroblastoma cell lines (Cell lines, n = 12), and neuroblastoma tumour tissue (NB tissue, n = 24), using the average of pyrosequencing assays 01 and 02; full results in C; *p
    Figure Legend Snippet: GATA3 DNA methylation in neuroblastoma. ( A ) GATA3 DNA methylation detected by MCIP. Black bars show the probe ratios derived from MCIP for hNCC and four neuroblastoma cell lines, positioned on the GATA3 CpG island promoter region, showing the sense and antisense transcripts and CpG island (CGI) (human genome build NCBI36/Hg18 visualised on the UCSC genome browser; http://genome.ucsc.edu ). The positions of the hypomethylated region and the two pyrosequencing assays (01 and 02) are shown in red at the top. ( B ) Dotboxplot of GATA3 antisense DNA methylation measured by pyrosequencing in normal tissues (NT, n = 4), neuroblastoma cell lines (Cell lines, n = 12), and neuroblastoma tumour tissue (NB tissue, n = 24), using the average of pyrosequencing assays 01 and 02; full results in C; *p

    Techniques Used: DNA Methylation Assay, Derivative Assay

    22) Product Images from "CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region"

    Article Title: CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region

    Journal:

    doi: 10.1158/0008-5472.CAN-08-1005

    BORIS, but not CTCF, DNA binding is methylation independent. A , BORIS, but not CTCF, binds to a methylated H19 DMR CTCF DNA-binding sequence. HCT116 cells were harvested and nuclear cell extracts were used for EMSA with a 32 P-labeled Sss I-methylated oligonucleotide
    Figure Legend Snippet: BORIS, but not CTCF, DNA binding is methylation independent. A , BORIS, but not CTCF, binds to a methylated H19 DMR CTCF DNA-binding sequence. HCT116 cells were harvested and nuclear cell extracts were used for EMSA with a 32 P-labeled Sss I-methylated oligonucleotide

    Techniques Used: Binding Assay, Methylation, Sequencing, Labeling

    23) Product Images from "Genetic and epigenetic variations contributed by Alu retrotransposition"

    Article Title: Genetic and epigenetic variations contributed by Alu retrotransposition

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-12-617

    PCR validation of putative Alu insertions (a-g) . The Alu insertions were sorted based on genomic coordinates. The Alu insertions were named AI1 through AI21. N-normal brain tissue DNA; E1, E2, and E3-brain tumor tissue (ependymoma) DNA from different individuals; P and R- ependymoma DNA, P is primary and R is relapsed tumor from the same individual.
    Figure Legend Snippet: PCR validation of putative Alu insertions (a-g) . The Alu insertions were sorted based on genomic coordinates. The Alu insertions were named AI1 through AI21. N-normal brain tissue DNA; E1, E2, and E3-brain tumor tissue (ependymoma) DNA from different individuals; P and R- ependymoma DNA, P is primary and R is relapsed tumor from the same individual.

    Techniques Used: Polymerase Chain Reaction

    24) Product Images from "SOXC transcription factors in mantle cell lymphoma: the role of promoter methylation in SOX11 expression"

    Article Title: SOXC transcription factors in mantle cell lymphoma: the role of promoter methylation in SOX11 expression

    Journal: Scientific Reports

    doi: 10.1038/srep01400

    The SOX11 promoter methylation level in MCL. (a) methylation level of SOX11 promoter in MCL cases, cell lines and non-malignant cells shown by the ΔCt values corresponding to the difference between McrBC-digested and non-digested DNA sequence of four fragments within the SOX11 promoter; the relative location of different fragments is presented beneath the chart on the color-coded graph, black block presents location of pyroprimers. (b) SOX11 promoter methylation shown by pyrosequencing. The Raji (Burkitt lymphoma) cell line with a hypermethylated SOX11 promoter was used as a control for high methylation.
    Figure Legend Snippet: The SOX11 promoter methylation level in MCL. (a) methylation level of SOX11 promoter in MCL cases, cell lines and non-malignant cells shown by the ΔCt values corresponding to the difference between McrBC-digested and non-digested DNA sequence of four fragments within the SOX11 promoter; the relative location of different fragments is presented beneath the chart on the color-coded graph, black block presents location of pyroprimers. (b) SOX11 promoter methylation shown by pyrosequencing. The Raji (Burkitt lymphoma) cell line with a hypermethylated SOX11 promoter was used as a control for high methylation.

    Techniques Used: Methylation, Sequencing, Blocking Assay

    25) Product Images from "Recurrent Chromosomal Copy Number Alterations in Sporadic Chordomas"

    Article Title: Recurrent Chromosomal Copy Number Alterations in Sporadic Chordomas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018846

    T quantitative real-time PCR. Two primer/probe sets were used to quantitate T (6q27) and MCM7 (7q21.3-q22.1) (n = 3). Relative T ∶ MCM7 ratios were normalized against an average ratio established from ten non-chordoma DNA samples (normal). The normalized ratios were corrected for MCM7 copy number from array CGH data and approximate tumor percentage based on histological review. Corrected normalized ratios were multiplied by 2 to obtain the absolute T copy number. The dashed line represents a normal copy number of 2.
    Figure Legend Snippet: T quantitative real-time PCR. Two primer/probe sets were used to quantitate T (6q27) and MCM7 (7q21.3-q22.1) (n = 3). Relative T ∶ MCM7 ratios were normalized against an average ratio established from ten non-chordoma DNA samples (normal). The normalized ratios were corrected for MCM7 copy number from array CGH data and approximate tumor percentage based on histological review. Corrected normalized ratios were multiplied by 2 to obtain the absolute T copy number. The dashed line represents a normal copy number of 2.

    Techniques Used: Real-time Polymerase Chain Reaction

    CDKN2A and PTEN methylation specific PCR. Bisulfite-treated chordoma DNA samples were tested with methylation specific PCR to evaluate for hypermethylation of the CDKN2A and PTEN promoter regions. Two sets of unmethylated (U) and methylated (M) PCR primers were used for each target gene (bottom labels, MSP1 and MSP2). Unmethylated and methylated controls are shown along with results for case CH33. Results for other tested cases are summarized in Tables 3 and 4 under the CDKN2A and PTEN MSP1 and MSP2 columns. Tick marks on the left and right of each panel indicate 100, 200, 300, and 400 base pair sizes (bottom to top).
    Figure Legend Snippet: CDKN2A and PTEN methylation specific PCR. Bisulfite-treated chordoma DNA samples were tested with methylation specific PCR to evaluate for hypermethylation of the CDKN2A and PTEN promoter regions. Two sets of unmethylated (U) and methylated (M) PCR primers were used for each target gene (bottom labels, MSP1 and MSP2). Unmethylated and methylated controls are shown along with results for case CH33. Results for other tested cases are summarized in Tables 3 and 4 under the CDKN2A and PTEN MSP1 and MSP2 columns. Tick marks on the left and right of each panel indicate 100, 200, 300, and 400 base pair sizes (bottom to top).

    Techniques Used: Methylation, Polymerase Chain Reaction

    26) Product Images from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation"

    Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

    Journal: Cancer Cell International

    doi: 10.1186/1475-2867-7-14

    Summary of Pyro-Q-CpG findings . Illustrated are the summary findings by Pyro-Q-CpG analysis of U2OS cells in vitro without treatment (control) and with 1 μM decitabine treatment (treated). The illustration also summarizes Pyro-Q-CpG findings in control U2OS xenograft tumors (Xeno-1, Xeno-2, and Xeno-3), and 2.5 mg/kg decitabine treated U2OS xenograft tumors (Xeno-4, Xeno-5, and Xeno-6). DNA from NHOst (normal low-passage human osteoblasts) was also analyzed for experiment control. DNAs from early embryonic DNA (Emb-DNA) and universally methylated DNA (Met-DNA) were used for negative and positive control respectively. CpG-islands are denoted by grey rectangles relative to the gene start site. The region further enlarged below corresponds to each tested CpG sequence. The tick marks denotes the individual CpG dinucleotides. The transcription start site is indicated by a directional arrow with the base pair numbers annotated for each tested sequence. The extent of methylation is represented by the scale bar (bottom right). GADD45A , PAWR , and PDCD5 , had a high level of methylation before decitabine treatment while HSPA9B had an intermediate level of methylation before treatment. In all cases the methylation was decreased significantly (p
    Figure Legend Snippet: Summary of Pyro-Q-CpG findings . Illustrated are the summary findings by Pyro-Q-CpG analysis of U2OS cells in vitro without treatment (control) and with 1 μM decitabine treatment (treated). The illustration also summarizes Pyro-Q-CpG findings in control U2OS xenograft tumors (Xeno-1, Xeno-2, and Xeno-3), and 2.5 mg/kg decitabine treated U2OS xenograft tumors (Xeno-4, Xeno-5, and Xeno-6). DNA from NHOst (normal low-passage human osteoblasts) was also analyzed for experiment control. DNAs from early embryonic DNA (Emb-DNA) and universally methylated DNA (Met-DNA) were used for negative and positive control respectively. CpG-islands are denoted by grey rectangles relative to the gene start site. The region further enlarged below corresponds to each tested CpG sequence. The tick marks denotes the individual CpG dinucleotides. The transcription start site is indicated by a directional arrow with the base pair numbers annotated for each tested sequence. The extent of methylation is represented by the scale bar (bottom right). GADD45A , PAWR , and PDCD5 , had a high level of methylation before decitabine treatment while HSPA9B had an intermediate level of methylation before treatment. In all cases the methylation was decreased significantly (p

    Techniques Used: In Vitro, Methylation, Positive Control, Sequencing

    27) Product Images from "Promoter Hypermethylation and Suppression of Glutathione Peroxidase 3 Are Associated with Inflammatory Breast Carcinogenesis"

    Article Title: Promoter Hypermethylation and Suppression of Glutathione Peroxidase 3 Are Associated with Inflammatory Breast Carcinogenesis

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2014/787195

    Gel electrophoresis of GPX3 methylation-specific PCR (GPX3-MSP) products. Representative results of MSP using (a) unmethylated primers and (b) methylated primers. M is the DNA marker; lanes N1 and N2 represent normal breast tissues, lanes 1–4 represent non-IBC carcinoma tissues, and lanes 4–8 represent IBC breast carcinoma tissues. (c) Bars represent intensity values of methylated/unmethylated (M/U) ratios as quantified by ImageJ software. Normal breast tissues ( n = 6) with an M/U ratio = 0 (no methylated bands were detected from MSP) were recognized as unmethylated. We detected a significant increase ( P = 0.04) in the M/U ratio in IBC ( n = 20) carcinoma tissues compared to non-IBC ( n = 20) carcinoma tissues.
    Figure Legend Snippet: Gel electrophoresis of GPX3 methylation-specific PCR (GPX3-MSP) products. Representative results of MSP using (a) unmethylated primers and (b) methylated primers. M is the DNA marker; lanes N1 and N2 represent normal breast tissues, lanes 1–4 represent non-IBC carcinoma tissues, and lanes 4–8 represent IBC breast carcinoma tissues. (c) Bars represent intensity values of methylated/unmethylated (M/U) ratios as quantified by ImageJ software. Normal breast tissues ( n = 6) with an M/U ratio = 0 (no methylated bands were detected from MSP) were recognized as unmethylated. We detected a significant increase ( P = 0.04) in the M/U ratio in IBC ( n = 20) carcinoma tissues compared to non-IBC ( n = 20) carcinoma tissues.

    Techniques Used: Nucleic Acid Electrophoresis, Methylation, Polymerase Chain Reaction, Marker, Software

    28) Product Images from "Silencing of TESTIN by dense biallelic promoter methylation is the most common molecular event in childhood acute lymphoblastic leukaemia"

    Article Title: Silencing of TESTIN by dense biallelic promoter methylation is the most common molecular event in childhood acute lymphoblastic leukaemia

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-163

    CoBRA of ALL bone marrow DNA . A . Diagram of reverse-strand bisulfite-specific PCR fragment showing location of potential Taq α I restriction sites (T). B . Taq α I digests of TES bisulfite-specific PCR products from normal PBL, ALL and remission samples. Fully methylated DNA will generate fragments of 161, 109, 26, 39 and 42 bp respectively. (Marker lane; 100, 200, 300, 400 and 500 bp bands; U - undigested PCR product).
    Figure Legend Snippet: CoBRA of ALL bone marrow DNA . A . Diagram of reverse-strand bisulfite-specific PCR fragment showing location of potential Taq α I restriction sites (T). B . Taq α I digests of TES bisulfite-specific PCR products from normal PBL, ALL and remission samples. Fully methylated DNA will generate fragments of 161, 109, 26, 39 and 42 bp respectively. (Marker lane; 100, 200, 300, 400 and 500 bp bands; U - undigested PCR product).

    Techniques Used: Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, Methylation, Marker

    Methylation plots for ALL bone marrow and normal PBL DNA . Bisulfite sequencing of ALL marrow, remission marrow and normal PBL samples; each horizontal grouping indicates one sequenced clone, individual circles represent each CpG site (open circles and closed circles represent unmethylated and methylated CpG sites respectively). A . Methylation plots of ALL samples initially assayed by MS-MLPA. B . Normal PBL DNA methylation plots. C . Further ALL methylation plots. D . Matched ALL and remission methylation plots. Dotted line indicates location of HhaI site used in MS-MLPA. Percent methylation is shown in parentheses.
    Figure Legend Snippet: Methylation plots for ALL bone marrow and normal PBL DNA . Bisulfite sequencing of ALL marrow, remission marrow and normal PBL samples; each horizontal grouping indicates one sequenced clone, individual circles represent each CpG site (open circles and closed circles represent unmethylated and methylated CpG sites respectively). A . Methylation plots of ALL samples initially assayed by MS-MLPA. B . Normal PBL DNA methylation plots. C . Further ALL methylation plots. D . Matched ALL and remission methylation plots. Dotted line indicates location of HhaI site used in MS-MLPA. Percent methylation is shown in parentheses.

    Techniques Used: Methylation, Methylation Sequencing, Mass Spectrometry, Multiplex Ligation-dependent Probe Amplification, DNA Methylation Assay

    29) Product Images from "Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle"

    Article Title: Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020881

    CpG methylation percentage of the RSVp after rAAV administration in NHP determined by high-throughput sequencing. NHP were injected IM (Mac 1, Mac 2, and Mac 9) or IV (Mac 10 and Mac 11) with the rAAV2/1-RSV-LEA29Y-WPRE-pA vector. Each animal received a dose of 5.10 12 vg/kg. Total DNA was extracted from transduced skeletal muscle (A) and liver (B) and subjected to a sodium bisulfite conversion and subsequent PCR amplification. 454 sequencing was performed to evaluate the Pyro1- and Pyro2-region methylation. (a) Bioinformatic analysis of this sample revealed some sequences for which all the CpGs were methylated. ( *** ) p value
    Figure Legend Snippet: CpG methylation percentage of the RSVp after rAAV administration in NHP determined by high-throughput sequencing. NHP were injected IM (Mac 1, Mac 2, and Mac 9) or IV (Mac 10 and Mac 11) with the rAAV2/1-RSV-LEA29Y-WPRE-pA vector. Each animal received a dose of 5.10 12 vg/kg. Total DNA was extracted from transduced skeletal muscle (A) and liver (B) and subjected to a sodium bisulfite conversion and subsequent PCR amplification. 454 sequencing was performed to evaluate the Pyro1- and Pyro2-region methylation. (a) Bioinformatic analysis of this sample revealed some sequences for which all the CpGs were methylated. ( *** ) p value

    Techniques Used: CpG Methylation Assay, Next-Generation Sequencing, Injection, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Sequencing, Methylation

    CpG methylation percentage of the RSVp after rAAV administration in NHP determined by low-throughput sequencing. NHP were injected IM (Mac 1, Mac 2, and Mac 9) or IV (Mac 10 and Mac 11) with the rAAV2/1-RSV-LEA29Y-WPRE-pA vector. Each animal received a dose of 5.10 12 vg/kg. Total DNA was extracted from transduced skeletal muscle (A) and liver (B) and subjected to sodium bisulfite conversion and subsequent PCR amplification. Each sample was read at least 4 times by PSQ96 pyrosequencing for the two CpG-rich plots Pyro1 and Pyro2. na: not analyzable. ( *** ) p value
    Figure Legend Snippet: CpG methylation percentage of the RSVp after rAAV administration in NHP determined by low-throughput sequencing. NHP were injected IM (Mac 1, Mac 2, and Mac 9) or IV (Mac 10 and Mac 11) with the rAAV2/1-RSV-LEA29Y-WPRE-pA vector. Each animal received a dose of 5.10 12 vg/kg. Total DNA was extracted from transduced skeletal muscle (A) and liver (B) and subjected to sodium bisulfite conversion and subsequent PCR amplification. Each sample was read at least 4 times by PSQ96 pyrosequencing for the two CpG-rich plots Pyro1 and Pyro2. na: not analyzable. ( *** ) p value

    Techniques Used: CpG Methylation Assay, Sequencing, Injection, Plasmid Preparation, Polymerase Chain Reaction, Amplification

    30) Product Images from "Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line"

    Article Title: Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103837

    Methylation status of individual CpG sites in the Tex13 promoter. A. The CpG islands of mouse Tex13 were predicted with the MethPrimer program ( http://www.urogene.org/ methprimer) and are indicated in gray. B. The DNA methylation status of individual CpG sites on the Tex13 promoter was assessed by sodium bisulfite sequencing analysis. Genomic DNA was prepared from F9 and NIH3T3 cells. The black and white circles represent methylated and unmethylated CpGs, respectively. C. Effect of methylation on Tex13 transcriptional activity. Luciferase constructs [pGL3-Basic and Tex13 (−402/+20)] were in vitro methylated or mock-methylated with Sss I methyltransferase, and transfected into F9 cells. Firefly luciferase activity was assessed and normalized with respect to that of Renilla luciferase. Data are shown as relative fold increases compared with the results from mock-methylated pGL3-Basic. The presented values represent the mean ± SD of three independent experiments; ** p
    Figure Legend Snippet: Methylation status of individual CpG sites in the Tex13 promoter. A. The CpG islands of mouse Tex13 were predicted with the MethPrimer program ( http://www.urogene.org/ methprimer) and are indicated in gray. B. The DNA methylation status of individual CpG sites on the Tex13 promoter was assessed by sodium bisulfite sequencing analysis. Genomic DNA was prepared from F9 and NIH3T3 cells. The black and white circles represent methylated and unmethylated CpGs, respectively. C. Effect of methylation on Tex13 transcriptional activity. Luciferase constructs [pGL3-Basic and Tex13 (−402/+20)] were in vitro methylated or mock-methylated with Sss I methyltransferase, and transfected into F9 cells. Firefly luciferase activity was assessed and normalized with respect to that of Renilla luciferase. Data are shown as relative fold increases compared with the results from mock-methylated pGL3-Basic. The presented values represent the mean ± SD of three independent experiments; ** p

    Techniques Used: Methylation, DNA Methylation Assay, Methylation Sequencing, Activity Assay, Luciferase, Construct, In Vitro, Transfection

    31) Product Images from "Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma"

    Article Title: Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

    Journal: Molecular Carcinogenesis

    doi: 10.1002/mc.22591

    MEGF10 DNA methylation in neuroblastoma. (A) MEGF10 methylation assayed by pyrosequencing assays 01_PM (unfilled bars) and 02_PM (black bars) in normal tissue (fetal muscle [FM], fetal kidney [FK], normal kidney [NK], fetal lung [FL], human neural crest cells [hNCC], fetal adrenal [FA]), neuroblastoma cell lines and neuroblastoma tumors, grouped by stage. (B) MEGF10 DNA methylation assayed by pyrosequencing assays 01_PM and 02_PM in hNCC and the four neuroblastoma cells lines used for MCIP. Bars show the percentage methylation at each CpG in the two pyrosequencing assays, positioned relative to the MEGF10 first exon and CpG island (CGI), using the UCSC genome browser ( http://genome.ucsc.edu ). (C) MEGF10 RNA expression assayed by QPCR (black bars) and DNA methylation levels detected by pyrosequencing (unfilled bars), in control tissues and neuroblastoma cell lines. RNA levels were normalized to the endogenous levels of TBP and expressed relative to universal RNA (full data in Figure S6). DNA methylation was calculated as the average of the 01_PM and 02_PM pyrosequencing assays (A). Neuroblastoma subtypes (I, N, or S) and MYCN amplification status are shown above the cell line names. (D) MEGF10 RNA expression in SHSY‐5Y cells treated with 2 μM azadC (AZA) for 2–4 d. RNA levels were normalized to the endogenous levels of TBP and expressed as fold expression relative to levels in controls (solvent‐treated). (E) BoxPlot of MEGF10 DNA methylation measured by pyrosequencing in normal tissues (NT, n = 6), neuroblastoma cell lines ( n = 9), and neuroblastoma tumor tissue (NB, n = 46), using the average of assays 01_PM and 02_PM; full results are shown in (A) (** P
    Figure Legend Snippet: MEGF10 DNA methylation in neuroblastoma. (A) MEGF10 methylation assayed by pyrosequencing assays 01_PM (unfilled bars) and 02_PM (black bars) in normal tissue (fetal muscle [FM], fetal kidney [FK], normal kidney [NK], fetal lung [FL], human neural crest cells [hNCC], fetal adrenal [FA]), neuroblastoma cell lines and neuroblastoma tumors, grouped by stage. (B) MEGF10 DNA methylation assayed by pyrosequencing assays 01_PM and 02_PM in hNCC and the four neuroblastoma cells lines used for MCIP. Bars show the percentage methylation at each CpG in the two pyrosequencing assays, positioned relative to the MEGF10 first exon and CpG island (CGI), using the UCSC genome browser ( http://genome.ucsc.edu ). (C) MEGF10 RNA expression assayed by QPCR (black bars) and DNA methylation levels detected by pyrosequencing (unfilled bars), in control tissues and neuroblastoma cell lines. RNA levels were normalized to the endogenous levels of TBP and expressed relative to universal RNA (full data in Figure S6). DNA methylation was calculated as the average of the 01_PM and 02_PM pyrosequencing assays (A). Neuroblastoma subtypes (I, N, or S) and MYCN amplification status are shown above the cell line names. (D) MEGF10 RNA expression in SHSY‐5Y cells treated with 2 μM azadC (AZA) for 2–4 d. RNA levels were normalized to the endogenous levels of TBP and expressed as fold expression relative to levels in controls (solvent‐treated). (E) BoxPlot of MEGF10 DNA methylation measured by pyrosequencing in normal tissues (NT, n = 6), neuroblastoma cell lines ( n = 9), and neuroblastoma tumor tissue (NB, n = 46), using the average of assays 01_PM and 02_PM; full results are shown in (A) (** P

    Techniques Used: DNA Methylation Assay, Methylation, RNA Expression, Real-time Polymerase Chain Reaction, Amplification, Expressing

    32) Product Images from "Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells"

    Article Title: Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0188052

    Absolute telomere length measurement of hADSCs in the presence of different concentration of ZnSO 4 for 48 hours of incubation. Cells were seeded at a density of 5×10 4 cells/wells for about 48 hours in the presence of 1.5×10 −8 and 2.99×10 −10 M ZnSO 4 . Following, Genomic DNA was isolated, telomere and single copy gene standard curve was created. Real-time PCR technique was used to measure the absolute telomere length. The data were analyzed as kb/reaction and the genome copies/reaction for the telomere and the SCG. As described in results section, 1.5×10 −8 M ZnSO 4 were significantly increased the telomere length of hADSCs (**P
    Figure Legend Snippet: Absolute telomere length measurement of hADSCs in the presence of different concentration of ZnSO 4 for 48 hours of incubation. Cells were seeded at a density of 5×10 4 cells/wells for about 48 hours in the presence of 1.5×10 −8 and 2.99×10 −10 M ZnSO 4 . Following, Genomic DNA was isolated, telomere and single copy gene standard curve was created. Real-time PCR technique was used to measure the absolute telomere length. The data were analyzed as kb/reaction and the genome copies/reaction for the telomere and the SCG. As described in results section, 1.5×10 −8 M ZnSO 4 were significantly increased the telomere length of hADSCs (**P

    Techniques Used: Concentration Assay, Incubation, Isolation, Real-time Polymerase Chain Reaction

    33) Product Images from "Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells"

    Article Title: Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.08.013

    DNMT3A R882H/C mutants impair apoptosis through attenuation of DNA damage signaling. (A) Cell proliferation of stably transduced U937 cells treated with 300 nM ATRA and 300 nM ABT-263 for 72 hours or no drug. Data presented were the average of at least two replicates. (B) Representative flow cytometry analysis (left panel) and the % of Annexin V– and PI-positive cells (right panel) of mutant and WT-DNMT3A U937 cells including EV on the treatment of 300 nM ABT-263 for 72 hours are shown. (C and D) DNA damage signaling protein levels including c-MYC were examined with or without treatment of ATRA (C) or ABT-263 (D) by immunoblot analyses. β-Actin was used as a control for equal loading. (E and F) Phosphorylation of H2A.X (γ-H2A.X) levels was verified in transformed U937 cells without drug (E) and in the presence of 300 nM ATRA (F) by immunofluorescence microscopy (original magnification: ×1000). All the images were taken with same contrast and exposure time. Quantitation of the intensity of γ-H2A.X per cell was measured using ImageJ software (NIH, USA). Each data point represents the mean ± S.D. of three different microscopic field. All studies were repeated at least once; * P
    Figure Legend Snippet: DNMT3A R882H/C mutants impair apoptosis through attenuation of DNA damage signaling. (A) Cell proliferation of stably transduced U937 cells treated with 300 nM ATRA and 300 nM ABT-263 for 72 hours or no drug. Data presented were the average of at least two replicates. (B) Representative flow cytometry analysis (left panel) and the % of Annexin V– and PI-positive cells (right panel) of mutant and WT-DNMT3A U937 cells including EV on the treatment of 300 nM ABT-263 for 72 hours are shown. (C and D) DNA damage signaling protein levels including c-MYC were examined with or without treatment of ATRA (C) or ABT-263 (D) by immunoblot analyses. β-Actin was used as a control for equal loading. (E and F) Phosphorylation of H2A.X (γ-H2A.X) levels was verified in transformed U937 cells without drug (E) and in the presence of 300 nM ATRA (F) by immunofluorescence microscopy (original magnification: ×1000). All the images were taken with same contrast and exposure time. Quantitation of the intensity of γ-H2A.X per cell was measured using ImageJ software (NIH, USA). Each data point represents the mean ± S.D. of three different microscopic field. All studies were repeated at least once; * P

    Techniques Used: Stable Transfection, Flow Cytometry, Cytometry, Mutagenesis, Transformation Assay, Immunofluorescence, Microscopy, Quantitation Assay, Software

    DNMT3A -R882 mutants deregulate cell cycle and apoptosis-associated genes in U937 cells. (A) Heatmap representation of cell-cycle facilitator and apoptosis-inhibiting genes identified as being differentially expressed in U937 cells transduced with DNMT3A -WT and mutants. Red indicates upregulated genes compared to WT (green). (B) Heatmap for gene expression in U937 cells transduced with DNMT3A -WT and mutants. Genes associated with cell-cycle inhibiting and apoptosis facilitators are clustered. Green indicates downregulated genes compared to WT- (red). (C and D) Quantitative RT-PCR results for 10 genes involved in cell cycle regulation and apoptosis (C) and representative genes associated with hematopoietic malignancies (D) showing the same patterns observed in gene-expression microarray analysis of WT- and mutants transduced U937 cells. (E) GO analyses of upregulated genes in R882C/H-expressing U937 cells showing a series of genes enriched in different cellular and molecular processes including cell cycle progression and DNA damage. Error bars represent ± S.D. of the mean of three to five experiments. * P ≤ .05, ** P ≤ .01, *** P ≤ .005 compared to EV.
    Figure Legend Snippet: DNMT3A -R882 mutants deregulate cell cycle and apoptosis-associated genes in U937 cells. (A) Heatmap representation of cell-cycle facilitator and apoptosis-inhibiting genes identified as being differentially expressed in U937 cells transduced with DNMT3A -WT and mutants. Red indicates upregulated genes compared to WT (green). (B) Heatmap for gene expression in U937 cells transduced with DNMT3A -WT and mutants. Genes associated with cell-cycle inhibiting and apoptosis facilitators are clustered. Green indicates downregulated genes compared to WT- (red). (C and D) Quantitative RT-PCR results for 10 genes involved in cell cycle regulation and apoptosis (C) and representative genes associated with hematopoietic malignancies (D) showing the same patterns observed in gene-expression microarray analysis of WT- and mutants transduced U937 cells. (E) GO analyses of upregulated genes in R882C/H-expressing U937 cells showing a series of genes enriched in different cellular and molecular processes including cell cycle progression and DNA damage. Error bars represent ± S.D. of the mean of three to five experiments. * P ≤ .05, ** P ≤ .01, *** P ≤ .005 compared to EV.

    Techniques Used: Transduction, Expressing, Quantitative RT-PCR, Microarray

    34) Product Images from "Genomic imprinting is variably lost during reprogramming of mouse iPS cells"

    Article Title: Genomic imprinting is variably lost during reprogramming of mouse iPS cells

    Journal: Stem cell research

    doi: 10.1016/j.scr.2013.05.011

    Maternally inherited DNA methylation imprint was variably lost at the Zac1 DMR region in the iPS clones. Bacterial colony sequencing of the bisulfite PCR product was performed for genomic DNA samples from the parental cells as well as from the iPS clones at early passages (
    Figure Legend Snippet: Maternally inherited DNA methylation imprint was variably lost at the Zac1 DMR region in the iPS clones. Bacterial colony sequencing of the bisulfite PCR product was performed for genomic DNA samples from the parental cells as well as from the iPS clones at early passages (

    Techniques Used: DNA Methylation Assay, Clone Assay, Sequencing, Polymerase Chain Reaction

    COBRA analysis of paternally inherited DNA methylation imprint. Genomic DNA was isolated from the iPS clones (Lanes 5–15), mouse tail of the mother (Lane 1) and father (Lane 2), MEF cells without doxycycline (Lane 3), MEF cells with doxycycline (Lane 4) as well as from the wild-type TC1 ES cells (Lane 16). COBRA was performed for three DMR regions (IG-DMR of the Dlk1-Dio3 imprinted region, H19 DMR, Rasgrf1 DMR). The iPS clones used for COBRA are: L7 (Lane 5), L9 (Lane 6), B11 (Lane 7), E7 (Lane 8), D15D (Lane 9), D36 (Lane 10), C8D (Lane 11), E7D (Lane 12), S4D (Lane 13), S5D (Lane 14) and n2 (Lane 15). U, unmethylated product. M, methylated product. A, IG-DMR with Taq I digestion. B, H19 DMR with Cla I digestion. C, Rasgrf1 DMR with Bst UI digestion.
    Figure Legend Snippet: COBRA analysis of paternally inherited DNA methylation imprint. Genomic DNA was isolated from the iPS clones (Lanes 5–15), mouse tail of the mother (Lane 1) and father (Lane 2), MEF cells without doxycycline (Lane 3), MEF cells with doxycycline (Lane 4) as well as from the wild-type TC1 ES cells (Lane 16). COBRA was performed for three DMR regions (IG-DMR of the Dlk1-Dio3 imprinted region, H19 DMR, Rasgrf1 DMR). The iPS clones used for COBRA are: L7 (Lane 5), L9 (Lane 6), B11 (Lane 7), E7 (Lane 8), D15D (Lane 9), D36 (Lane 10), C8D (Lane 11), E7D (Lane 12), S4D (Lane 13), S5D (Lane 14) and n2 (Lane 15). U, unmethylated product. M, methylated product. A, IG-DMR with Taq I digestion. B, H19 DMR with Cla I digestion. C, Rasgrf1 DMR with Bst UI digestion.

    Techniques Used: Combined Bisulfite Restriction Analysis Assay, DNA Methylation Assay, Isolation, Clone Assay, Methylation

    COBRA analysis of maternally inherited DNA methylation imprint. Genomic DNA was isolated from the iPS clones (Lanes 5–15 for Fig. 3A–C and Lanes 5–16 for Fig. 3D–E), mouse tail of the mother (Lane 1) and father (Lane 2), MEF cells without doxycycline (Lane 3), MEF cells with doxycycline (Lane 4) as well as from the wild-type TC1 ES cells (Lane 16 for A, B and C, Lane 17 for D and E). COBRA was performed for five DMR regions ( Peg1 , Peg3 , Snrpn , Zac1 and Peg10 ). The iPS clones used for COBRA are: L7 (Lane 5), L9 (Lane 6), B11 (Lane 7), E7 (Lane 8), D15D (Lane 9), D36 (Lane 10), C8D (Lane 11), E7D (Lane 12), S4D (Lane 13), S5D (Lane 14), n2 (Lane 15) and DL3 (Lane 16 for D and E). U, unmethylated product. M, methylated product. A, Peg1 DMR with Cla I digestion. B, Peg3 DMR with Taq I digestion. C, Snrpn DMR with Hha I digestion. D, Zac1 DMR with Taq I digestion. E, Peg10 DMR with Hha I digestion.
    Figure Legend Snippet: COBRA analysis of maternally inherited DNA methylation imprint. Genomic DNA was isolated from the iPS clones (Lanes 5–15 for Fig. 3A–C and Lanes 5–16 for Fig. 3D–E), mouse tail of the mother (Lane 1) and father (Lane 2), MEF cells without doxycycline (Lane 3), MEF cells with doxycycline (Lane 4) as well as from the wild-type TC1 ES cells (Lane 16 for A, B and C, Lane 17 for D and E). COBRA was performed for five DMR regions ( Peg1 , Peg3 , Snrpn , Zac1 and Peg10 ). The iPS clones used for COBRA are: L7 (Lane 5), L9 (Lane 6), B11 (Lane 7), E7 (Lane 8), D15D (Lane 9), D36 (Lane 10), C8D (Lane 11), E7D (Lane 12), S4D (Lane 13), S5D (Lane 14), n2 (Lane 15) and DL3 (Lane 16 for D and E). U, unmethylated product. M, methylated product. A, Peg1 DMR with Cla I digestion. B, Peg3 DMR with Taq I digestion. C, Snrpn DMR with Hha I digestion. D, Zac1 DMR with Taq I digestion. E, Peg10 DMR with Hha I digestion.

    Techniques Used: Combined Bisulfite Restriction Analysis Assay, DNA Methylation Assay, Isolation, Clone Assay, Methylation

    Maternally inherited DNA methylation imprint was variably lost at the Snrpn DMR in iPS clones. Bacterial colony sequencing of the bisulfite PCR product was performed for genomic DNA samples from the parental cells as well as from the iPS clones at early passages (
    Figure Legend Snippet: Maternally inherited DNA methylation imprint was variably lost at the Snrpn DMR in iPS clones. Bacterial colony sequencing of the bisulfite PCR product was performed for genomic DNA samples from the parental cells as well as from the iPS clones at early passages (

    Techniques Used: DNA Methylation Assay, Clone Assay, Sequencing, Polymerase Chain Reaction

    35) Product Images from "DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA"

    Article Title: DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-017-0409-7

    False positive exclusion of MeDIP-seq by MB-seq. a The distribution of sequencing depth across different methylation levels in MB-seq. b The distribution of the density of methylation sites across different methylation levels in MB-seq. All information was obtained using a 200 bp window on the genome-wide level. c The distribution of read depth, density of methylation sites in MB-seq and methylation level of 5mC in BS-seq across a randomly genomic region. d Zooming in to a specific region, the red box shows a captured region with no methylated sites, which were nonspecific DNA fragments captured by 5’methylcytosine antibody. e The percentage of windows (200 bp) with less certain methylation sites
    Figure Legend Snippet: False positive exclusion of MeDIP-seq by MB-seq. a The distribution of sequencing depth across different methylation levels in MB-seq. b The distribution of the density of methylation sites across different methylation levels in MB-seq. All information was obtained using a 200 bp window on the genome-wide level. c The distribution of read depth, density of methylation sites in MB-seq and methylation level of 5mC in BS-seq across a randomly genomic region. d Zooming in to a specific region, the red box shows a captured region with no methylated sites, which were nonspecific DNA fragments captured by 5’methylcytosine antibody. e The percentage of windows (200 bp) with less certain methylation sites

    Techniques Used: Methylated DNA Immunoprecipitation, Sequencing, Methylation, Genome Wide

    36) Product Images from "Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes"

    Article Title: Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047942

    Effect of non-specific gDNA on the specificity and sensitivity of MSP. The unmethylated Ins2 gene plasmid was diluted in the presence or absence of 500 ng non-specific gDNA, bisulfite-treated, and used as template for PCR. A) 160, 80, 40, 20, 10, 5, and 2 copies of plasmid without non-specific DNA analyzed by qMSP using primer set P4/P6. B) Serial dilutions ranged from 10 8 to 10 copies of plasmid in the presence of non-specific gDNA analyzed by qMSP using primer set P4/P6. C) Serial dilution from 10 8 to 3 copies of plasmid in the absence of non-specific gDNA analyzed by qMSP using primer set P12/P13. D) Range of serial dilutions from 10 7 to 10 copies plasmid in the presence of non-specific gDNA analyzed by qMSP using primer set P12/P13. Mouse liver gDNA was used as non-specific DNA and NTC is the non-template control.
    Figure Legend Snippet: Effect of non-specific gDNA on the specificity and sensitivity of MSP. The unmethylated Ins2 gene plasmid was diluted in the presence or absence of 500 ng non-specific gDNA, bisulfite-treated, and used as template for PCR. A) 160, 80, 40, 20, 10, 5, and 2 copies of plasmid without non-specific DNA analyzed by qMSP using primer set P4/P6. B) Serial dilutions ranged from 10 8 to 10 copies of plasmid in the presence of non-specific gDNA analyzed by qMSP using primer set P4/P6. C) Serial dilution from 10 8 to 3 copies of plasmid in the absence of non-specific gDNA analyzed by qMSP using primer set P12/P13. D) Range of serial dilutions from 10 7 to 10 copies plasmid in the presence of non-specific gDNA analyzed by qMSP using primer set P12/P13. Mouse liver gDNA was used as non-specific DNA and NTC is the non-template control.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Serial Dilution

    Quantification of circulating beta cell DNA in STZ-treated diabetic mice. NOD/scid mice were injected with STZ at days 0, 1, and 2, and blood was collected pre-treatment and post-treatment days 1, 2, 3, 5, 6, 7, 14, and 35. A) Blood glucose levels for untreated (n = 3) and STZ-injected (n = 34) NOD/scid mice were measured at days 1 (n = 6), 2 (n = 4), 5 (n = 4), 6 (n = 8), 7 (n = 4), 14 (n = 4) and 35 (n = 4) after injection. In parallel, qMSP was done using bisulfite converted gDNA obtained from the blood of untreated (n = 3) and STZ-treated mice at designated time points. Fold changes in demethylation are measured by calculation of ΔΔC q (B), Relative Expression Ration (C) or Demethylation Index (D) for each sample using methylation-specific primers P12/P13 and bisulfite-specific primers P16/P17. The cloned Ins2 gene was used for normalization and standardization of the results as described under Material and Methods. The data display the mean ± standard error (SEM) of three independent measurements. The statistical significance was calculated with the Student t test for unpaired values and significance level indicated by asterisks (*, P
    Figure Legend Snippet: Quantification of circulating beta cell DNA in STZ-treated diabetic mice. NOD/scid mice were injected with STZ at days 0, 1, and 2, and blood was collected pre-treatment and post-treatment days 1, 2, 3, 5, 6, 7, 14, and 35. A) Blood glucose levels for untreated (n = 3) and STZ-injected (n = 34) NOD/scid mice were measured at days 1 (n = 6), 2 (n = 4), 5 (n = 4), 6 (n = 8), 7 (n = 4), 14 (n = 4) and 35 (n = 4) after injection. In parallel, qMSP was done using bisulfite converted gDNA obtained from the blood of untreated (n = 3) and STZ-treated mice at designated time points. Fold changes in demethylation are measured by calculation of ΔΔC q (B), Relative Expression Ration (C) or Demethylation Index (D) for each sample using methylation-specific primers P12/P13 and bisulfite-specific primers P16/P17. The cloned Ins2 gene was used for normalization and standardization of the results as described under Material and Methods. The data display the mean ± standard error (SEM) of three independent measurements. The statistical significance was calculated with the Student t test for unpaired values and significance level indicated by asterisks (*, P

    Techniques Used: Mouse Assay, Injection, Expressing, Methylation, Clone Assay

    37) Product Images from "Estrogen-mediated epigenetic repression of large chromosomal regions through DNA looping"

    Article Title: Estrogen-mediated epigenetic repression of large chromosomal regions through DNA looping

    Journal: Genome Research

    doi: 10.1101/gr.101923.109

    Genome-wide mapping of DNA methylation in MCF-7 breast cancer cells. MCF-7 cells were stimulated with E2 for 4 h and then subjected to MeDIP-chip. Combined with Pol II ChIP-seq data of MDECs and MCF-7 cells, 11 hypermethylated zones, as indicated by the
    Figure Legend Snippet: Genome-wide mapping of DNA methylation in MCF-7 breast cancer cells. MCF-7 cells were stimulated with E2 for 4 h and then subjected to MeDIP-chip. Combined with Pol II ChIP-seq data of MDECs and MCF-7 cells, 11 hypermethylated zones, as indicated by the

    Techniques Used: Genome Wide, DNA Methylation Assay, Methylated DNA Immunoprecipitation, Chromatin Immunoprecipitation

    38) Product Images from "Identification of DNA methylation-independent epigenetic events underlying clear cell renal cell carcinoma"

    Article Title: Identification of DNA methylation-independent epigenetic events underlying clear cell renal cell carcinoma

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-15-2622

    Overview of Acce SssI ble data in ccRCC tumor/normal tissue pairs A) Δβ-values of no-enzyme-treated (endogenous methylation) and M. Sss I-treated (accessibility) samples were plotted to visualize DNA methylation and accessibility changes simultaneously. Bar plots quantify these changes and illustrate the distribution of CpG-islands and probe location. B) Six groups of epigenetic changes analyzed: Group a , loss of chromatin accessibility but gain in DNA methylation; Group b , no change in chromatin accessibility but gain in DNA methylation; Group c , no change in chromatin accessibility but loss of DNA methylation; Group d , gain in DNA accessibility but loss of DNA methylation; Group e , loss of chromatin accessibility but no change in DNA methylation; Group f , gain in chromatin accessibility but no change in DNA methylation.
    Figure Legend Snippet: Overview of Acce SssI ble data in ccRCC tumor/normal tissue pairs A) Δβ-values of no-enzyme-treated (endogenous methylation) and M. Sss I-treated (accessibility) samples were plotted to visualize DNA methylation and accessibility changes simultaneously. Bar plots quantify these changes and illustrate the distribution of CpG-islands and probe location. B) Six groups of epigenetic changes analyzed: Group a , loss of chromatin accessibility but gain in DNA methylation; Group b , no change in chromatin accessibility but gain in DNA methylation; Group c , no change in chromatin accessibility but loss of DNA methylation; Group d , gain in DNA accessibility but loss of DNA methylation; Group e , loss of chromatin accessibility but no change in DNA methylation; Group f , gain in chromatin accessibility but no change in DNA methylation.

    Techniques Used: Methylation, DNA Methylation Assay

    Diagram of Acce SssI ble assay Tissues were obtained and processed as described in Materials and Methods. Tissues were processed and nuclei were either left untreated or were treated with the M. Sss I methyltransferase enzyme. Endogenously methylated loci are unable to be interrogated (as illustrated). Following bisulfite conversion and HM450 analyses, untreated (endogenous) DNA methylation values from each sample were subtracted from respective enzyme-treated (exogenous) DNA methylation values, revealing chromatin accessibility changes.
    Figure Legend Snippet: Diagram of Acce SssI ble assay Tissues were obtained and processed as described in Materials and Methods. Tissues were processed and nuclei were either left untreated or were treated with the M. Sss I methyltransferase enzyme. Endogenously methylated loci are unable to be interrogated (as illustrated). Following bisulfite conversion and HM450 analyses, untreated (endogenous) DNA methylation values from each sample were subtracted from respective enzyme-treated (exogenous) DNA methylation values, revealing chromatin accessibility changes.

    Techniques Used: Methylation, DNA Methylation Assay

    39) Product Images from "Retrotransposon-based profiling of mammalian epigenomes: DNA methylation of IAP LTRs in embryonic stem, somatic and cancer cells"

    Article Title: Retrotransposon-based profiling of mammalian epigenomes: DNA methylation of IAP LTRs in embryonic stem, somatic and cancer cells

    Journal: Genomics

    doi: 10.1016/j.ygeno.2014.09.009

    DNA methylation profiles of IAP LTRs in ES and Neuro2A cells compared to somatic cells
    Figure Legend Snippet: DNA methylation profiles of IAP LTRs in ES and Neuro2A cells compared to somatic cells

    Techniques Used: DNA Methylation Assay

    40) Product Images from "Genome-wide DNA methylation study identifies genes associated with the cardiovascular biomarker GDF-15"

    Article Title: Genome-wide DNA methylation study identifies genes associated with the cardiovascular biomarker GDF-15

    Journal: Human molecular genetics

    doi: 10.1093/hmg/ddv511

    Regional plot of the association between GDF-15 levels and DNA methylation in the VNP1/MIR21 region. Both genes are coded on the positive strand with the transcription-starting site being located at the most left part of respective gene. The black marks is the –log10( P -values) for the association in the NSPHS and the grey marks in the PIVUS study.
    Figure Legend Snippet: Regional plot of the association between GDF-15 levels and DNA methylation in the VNP1/MIR21 region. Both genes are coded on the positive strand with the transcription-starting site being located at the most left part of respective gene. The black marks is the –log10( P -values) for the association in the NSPHS and the grey marks in the PIVUS study.

    Techniques Used: DNA Methylation Assay

    41) Product Images from "The tobacco carcinogen NNK drives accumulation of DNMT1 at the GR promoter thereby reducing GR expression in untransformed lung fibroblasts"

    Article Title: The tobacco carcinogen NNK drives accumulation of DNMT1 at the GR promoter thereby reducing GR expression in untransformed lung fibroblasts

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23309-2

    Long term exposure of MRC-5 cells to NNK results in a decrease in GRα and an increase in DNMT1 expression. Exposure to the GR antagonist, RU486, has the same effect. Clonogenic and comet analysis reveal that NNK has no effect on cell proliferation while RU486 enhances proliferation. By 106 days, NNK induces significant DNA damage. ( A ) Relative expression of GRα and DNMT1 in MRC-5 cells treated with either NNK or RU486 for 62 days (n = 3, p = 0.001 compared to the control). ( B ) Clonogenic assay performed on cells chronically treated with either NNK or RU486 for 86 days. ( C ) Comet assay showing extent of DNA damage after 29 and 106 days of chronic NNK or RU486 treatment (n = 300, p
    Figure Legend Snippet: Long term exposure of MRC-5 cells to NNK results in a decrease in GRα and an increase in DNMT1 expression. Exposure to the GR antagonist, RU486, has the same effect. Clonogenic and comet analysis reveal that NNK has no effect on cell proliferation while RU486 enhances proliferation. By 106 days, NNK induces significant DNA damage. ( A ) Relative expression of GRα and DNMT1 in MRC-5 cells treated with either NNK or RU486 for 62 days (n = 3, p = 0.001 compared to the control). ( B ) Clonogenic assay performed on cells chronically treated with either NNK or RU486 for 86 days. ( C ) Comet assay showing extent of DNA damage after 29 and 106 days of chronic NNK or RU486 treatment (n = 300, p

    Techniques Used: Expressing, Clonogenic Assay, Single Cell Gel Electrophoresis

    42) Product Images from "Rhinovirus infections change DNA methylation and mRNA expression in children with asthma"

    Article Title: Rhinovirus infections change DNA methylation and mRNA expression in children with asthma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0205275

    RIDM of cg24890294 in BAT3 in nasal epithelial cells of asthmatic and controls. Changes in DNA methylation were detected with a HumanMethylation450 BeadChip Kit (A), verified by pyrosequencing (B), and modified mRNA expression (ΔHRVI-MOCK) detected by RNA sequencing (C) and qPCR (D). The correlation between change in DNA methylation of cg24890294 and change in mRNA expression of BAT3 (E). Crosses represent children with asthma and the triangles represent controls.
    Figure Legend Snippet: RIDM of cg24890294 in BAT3 in nasal epithelial cells of asthmatic and controls. Changes in DNA methylation were detected with a HumanMethylation450 BeadChip Kit (A), verified by pyrosequencing (B), and modified mRNA expression (ΔHRVI-MOCK) detected by RNA sequencing (C) and qPCR (D). The correlation between change in DNA methylation of cg24890294 and change in mRNA expression of BAT3 (E). Crosses represent children with asthma and the triangles represent controls.

    Techniques Used: DNA Methylation Assay, Modification, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    43) Product Images from "Germline Allele-Specific Expression of DAPK1 in Chronic Lymphocytic Leukemia"

    Article Title: Germline Allele-Specific Expression of DAPK1 in Chronic Lymphocytic Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055261

    Allele-specific DNA methylation (ASM) contributes to ASE of DAPK1 in Granta-519 cells. (A) Scheme of the DAPK1 promoter region and the associated CpG island. Grey boxes display the first 2 exons of DAPK1 . Nucleotide positions are given relative to DAPK1 transcriptional start site (TSS). Dashed lines represent positions of the investigated regions/amplicons. (B) Quantitative DNA methylation analysis of the DAPK1 gene 5′ region (amplicons A–E) in untreated and 5-aza-2′-deoxycytidine (DAC)-treated Granta-519 cells was performed using the MassARRAY-based MassCleave method. Bars represent quantitative DNA methylation values (%) at single CpG units. (C) Bisulfite-sequencing of the DAPK1 5′ region including the SNP rs13300553 (G/A) used for allelic separation in Granta-519 cells. Sequenced clones carrying A at the respective SNP site (+520) are grouped in the upper panel, the G alleles are displayed in the lower panel. Black boxes represent single-CpG methylation, grey boxes represent unmethylated CpGs, white boxes stand for missing data. Methylation levels are calculated over the area between +58 and +263 in both allele groups. (D) Detection of ASM by separate amplification of either the unmethylated or methylated alleles by methylation-specific PCR on bisulfite-converted genomic DNA. Genotype distribution between the differentially methylated alleles was performed by SNuPE/MALDI-TOF. Untreated Granta-519 (PBS), 7-day treatment with the DNMT inhibitor 5-aza-2′-deoxycytidine (DAC), and assessment of ASM after 33 days of withdrawal of DAC (33-day recovery) are shown. The right panel shows the assessment of a CpG dinucleotide as specificity control (see results section for detailed explanation).
    Figure Legend Snippet: Allele-specific DNA methylation (ASM) contributes to ASE of DAPK1 in Granta-519 cells. (A) Scheme of the DAPK1 promoter region and the associated CpG island. Grey boxes display the first 2 exons of DAPK1 . Nucleotide positions are given relative to DAPK1 transcriptional start site (TSS). Dashed lines represent positions of the investigated regions/amplicons. (B) Quantitative DNA methylation analysis of the DAPK1 gene 5′ region (amplicons A–E) in untreated and 5-aza-2′-deoxycytidine (DAC)-treated Granta-519 cells was performed using the MassARRAY-based MassCleave method. Bars represent quantitative DNA methylation values (%) at single CpG units. (C) Bisulfite-sequencing of the DAPK1 5′ region including the SNP rs13300553 (G/A) used for allelic separation in Granta-519 cells. Sequenced clones carrying A at the respective SNP site (+520) are grouped in the upper panel, the G alleles are displayed in the lower panel. Black boxes represent single-CpG methylation, grey boxes represent unmethylated CpGs, white boxes stand for missing data. Methylation levels are calculated over the area between +58 and +263 in both allele groups. (D) Detection of ASM by separate amplification of either the unmethylated or methylated alleles by methylation-specific PCR on bisulfite-converted genomic DNA. Genotype distribution between the differentially methylated alleles was performed by SNuPE/MALDI-TOF. Untreated Granta-519 (PBS), 7-day treatment with the DNMT inhibitor 5-aza-2′-deoxycytidine (DAC), and assessment of ASM after 33 days of withdrawal of DAC (33-day recovery) are shown. The right panel shows the assessment of a CpG dinucleotide as specificity control (see results section for detailed explanation).

    Techniques Used: DNA Methylation Assay, Methylation Sequencing, Clone Assay, CpG Methylation Assay, Methylation, Amplification, Polymerase Chain Reaction

    Characterization of a multiplexed MassARRAY-based method for detection of allele-specific expression (ASE). (A) Representative MassARRAY spectra of molecular standards of DAPK1 exonic SNP rs1056719 (G/A). Each spectrum represents the mass range from 5390 to 5500 Da displaying SNP rs1056719 out of a multiplexed assay. Left peaks represent the G allele, right peaks represent the A allele. Copy number ratios between standard plasmids containing G and A alleles are given below each spectrum. (B) Standard curves for a plasmid-based standard displaying allelic ratios from 1∶50 to 50∶1 and correlation with idealized ratio (SNP rs1056719). The correlation was calculated using the Pearson correlation coefficient. (C) Standard curves for a genomic DNA based standard displaying allelic ratios from 1∶50 to 50∶1 and correlation with the idealized ratio (SNP rs1056719).
    Figure Legend Snippet: Characterization of a multiplexed MassARRAY-based method for detection of allele-specific expression (ASE). (A) Representative MassARRAY spectra of molecular standards of DAPK1 exonic SNP rs1056719 (G/A). Each spectrum represents the mass range from 5390 to 5500 Da displaying SNP rs1056719 out of a multiplexed assay. Left peaks represent the G allele, right peaks represent the A allele. Copy number ratios between standard plasmids containing G and A alleles are given below each spectrum. (B) Standard curves for a plasmid-based standard displaying allelic ratios from 1∶50 to 50∶1 and correlation with idealized ratio (SNP rs1056719). The correlation was calculated using the Pearson correlation coefficient. (C) Standard curves for a genomic DNA based standard displaying allelic ratios from 1∶50 to 50∶1 and correlation with the idealized ratio (SNP rs1056719).

    Techniques Used: Expressing, Plasmid Preparation

    44) Product Images from "DNMT1-interacting RNAs block gene specific DNA methylation"

    Article Title: DNMT1-interacting RNAs block gene specific DNA methylation

    Journal: Nature

    doi: 10.1038/nature12598

    Loss- and gain-of-function studies demonstrate that ecCEBPA maintains CEBPA expression by regulating methylation of the CEBPA locus a , Diagram indicates: position of target sequences for shRNA constructs (sh1–3); the fragment derived from the ecCEBPA employed for overexpression (R1) regions analyzed for changes in DNA methylation (distal promoter; coding sequence, CDS; and 3′UTR); b–c , The results of ecCEBPA loss-of-function in CEBPA -expressing U937 cells. Effect of ecCEBPA -targeting shRNAs on CEBPA mRNA levels. qRT-PCR, bars indicate mean ± s.d. ( b ) and methylation of the CEBPA promoter ( c ). DNA methylation changes are shown as the ratios of methylated to unmethylated CpGs in all clones analyzed per each construct (n=14); d–e , The results of ecCEBPA gain-of-function studies in K562 cells, in which CEBPA is methylated and silenced. d , Effect of ecCEBPA upregulation on CEBPA mRNA levels. UR = unrelated region. qRT-PCR, bars indicate mean ± s.d. (n=4); e , Effect of ecCEBPA upregulation on methylation of the CEBPA locus (DNA methylation changes were assessed as described in c ; (n=14, for distal promoter; and n=6, for CDS, 3′UTR); f–g , The results of transcription inhibition in U937 cells. f, ec CEBPA expression levels after treatment with Actinomycin D and ML-60218 in synchronized and unsynchronized cells. qRT-PCR, bars indicate mean ± s.d.; g , DNA methylation changes after treatment with Actinomycin D and ML-60218 in synchronized (n=12) and unsynchronized (n=10) cells (assessed as described in c) . All bisulfite sequenced clones were analyzed by Fisher’s exact test. * P
    Figure Legend Snippet: Loss- and gain-of-function studies demonstrate that ecCEBPA maintains CEBPA expression by regulating methylation of the CEBPA locus a , Diagram indicates: position of target sequences for shRNA constructs (sh1–3); the fragment derived from the ecCEBPA employed for overexpression (R1) regions analyzed for changes in DNA methylation (distal promoter; coding sequence, CDS; and 3′UTR); b–c , The results of ecCEBPA loss-of-function in CEBPA -expressing U937 cells. Effect of ecCEBPA -targeting shRNAs on CEBPA mRNA levels. qRT-PCR, bars indicate mean ± s.d. ( b ) and methylation of the CEBPA promoter ( c ). DNA methylation changes are shown as the ratios of methylated to unmethylated CpGs in all clones analyzed per each construct (n=14); d–e , The results of ecCEBPA gain-of-function studies in K562 cells, in which CEBPA is methylated and silenced. d , Effect of ecCEBPA upregulation on CEBPA mRNA levels. UR = unrelated region. qRT-PCR, bars indicate mean ± s.d. (n=4); e , Effect of ecCEBPA upregulation on methylation of the CEBPA locus (DNA methylation changes were assessed as described in c ; (n=14, for distal promoter; and n=6, for CDS, 3′UTR); f–g , The results of transcription inhibition in U937 cells. f, ec CEBPA expression levels after treatment with Actinomycin D and ML-60218 in synchronized and unsynchronized cells. qRT-PCR, bars indicate mean ± s.d.; g , DNA methylation changes after treatment with Actinomycin D and ML-60218 in synchronized (n=12) and unsynchronized (n=10) cells (assessed as described in c) . All bisulfite sequenced clones were analyzed by Fisher’s exact test. * P

    Techniques Used: Expressing, Methylation, shRNA, Construct, Derivative Assay, Over Expression, DNA Methylation Assay, Sequencing, Quantitative RT-PCR, Clone Assay, Inhibition

    ecCEBPA –DNMT1 interactions; DNMT1 binds to RNA with greater affinity than to DNA a , Diagram showing position of qRT-PCR primers used in RIP, double-headed arrow; RNA and DNA oligonucleotides used in EMSA and REMSA. Asterisks indicate position of methylated cytosines; umDNA, hmDNA, and mDNA refer to unmethylated, hemimethylated, and methylated DNA probes, respectively; b , ecCEBPA is immunoprecipitated with anti-DNMT1 antibody. qRT-PCR, bars indicate mean ± s.d.; c , RNA- DNMT1 binding is not affected by the absence of CpG dinucleotides (right panel). Left and middle panels: RNA oligonucleotide R2 and its mutated form mut R2 (asterisks indicate cytosines substituted into uridines), both able to form stem-loop-structures; d , RNA oligonucleotide able to form stem-loop structure bind DNMT1 (R6); e , R5 RNA oligonucleotide forming stem-loop structure (R5) has a greater DNMT1 affinity compared to mut R5, unable to fold into stem-loop, (taken in equimolar amounts), at the same DNMT1 concentration; f , Left four panels: REMSA and EMSA performed with the fixed concentration of ssRNA and dsDNA oligonucleotides (1 nM) and increasing concentrations of DNMT1 protein; Right panel: Nonlinear regression analysis of bound RNA/DNA versus DNMT1 concentrations. Error bars indicate s.d. from two independent experiments; g , REMSA showing that RNA oligonucleotide R4, which is unable to form stem-loop structure, displays lower DNMT1 affinity as compared to R5 (Fig. 3f left panel) at the same DNMT1 concentrations; h , Left panel: Schematic diagram showing the DNMT1 domains and the GST-DNMT1 isolated fragments (F1–F5); Right panel: GST-DNMT1 pull down assay demonstrating binding of the folded RNA oligonucleotide R5 to the catalytic domain of DNMT1.
    Figure Legend Snippet: ecCEBPA –DNMT1 interactions; DNMT1 binds to RNA with greater affinity than to DNA a , Diagram showing position of qRT-PCR primers used in RIP, double-headed arrow; RNA and DNA oligonucleotides used in EMSA and REMSA. Asterisks indicate position of methylated cytosines; umDNA, hmDNA, and mDNA refer to unmethylated, hemimethylated, and methylated DNA probes, respectively; b , ecCEBPA is immunoprecipitated with anti-DNMT1 antibody. qRT-PCR, bars indicate mean ± s.d.; c , RNA- DNMT1 binding is not affected by the absence of CpG dinucleotides (right panel). Left and middle panels: RNA oligonucleotide R2 and its mutated form mut R2 (asterisks indicate cytosines substituted into uridines), both able to form stem-loop-structures; d , RNA oligonucleotide able to form stem-loop structure bind DNMT1 (R6); e , R5 RNA oligonucleotide forming stem-loop structure (R5) has a greater DNMT1 affinity compared to mut R5, unable to fold into stem-loop, (taken in equimolar amounts), at the same DNMT1 concentration; f , Left four panels: REMSA and EMSA performed with the fixed concentration of ssRNA and dsDNA oligonucleotides (1 nM) and increasing concentrations of DNMT1 protein; Right panel: Nonlinear regression analysis of bound RNA/DNA versus DNMT1 concentrations. Error bars indicate s.d. from two independent experiments; g , REMSA showing that RNA oligonucleotide R4, which is unable to form stem-loop structure, displays lower DNMT1 affinity as compared to R5 (Fig. 3f left panel) at the same DNMT1 concentrations; h , Left panel: Schematic diagram showing the DNMT1 domains and the GST-DNMT1 isolated fragments (F1–F5); Right panel: GST-DNMT1 pull down assay demonstrating binding of the folded RNA oligonucleotide R5 to the catalytic domain of DNMT1.

    Techniques Used: Quantitative RT-PCR, Methylation, Immunoprecipitation, Binding Assay, Concentration Assay, Isolation, Pull Down Assay

    45) Product Images from "Promoter hypermethylation of the SFRP2 gene is a high-frequent alteration and tumor-specific epigenetic marker in human breast cancer"

    Article Title: Promoter hypermethylation of the SFRP2 gene is a high-frequent alteration and tumor-specific epigenetic marker in human breast cancer

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-7-83

    Global demethylation and histone acetylation restores SFRP2 expression . (A) MSP of four malignant cell lines was performed with DNA from either untreated cells, or after treatment with 1 μM DAC, or after treatment with 300 nM TSA, or after a combined treatment applying both drugs. In three cell lines (BT20, MCF7, T47D) a promoter demethylating effect could be visually detected, since signals indicative of unmethylated SFRP2 promoter arise (BT20, MCF7) or become enhanced (T47D) after the combined treatment. In T47D, DAC alone had no detectable demethylating effect on the SFRP2 promoter. (B) Expression of SFRP2 mRNA before treatment, or after treatment with 1 μM DAC, or after treatment with 300 nM TSA, or after a combined treatment applying both drugs. Treatment with DAC alone was not able to induce SFRP2 expression in all cell lines, in contrast to TSA which induced expression in two out of four cell lines (SKBR3 and T47D) previously showing partial SFRP2 methylation. However, only combined promoter demethylation and histone reacetylation leads to strong induction of SFRP2 mRNA expression in all cell lines. GAPDH served as cDNA loading control. (C) Suppression of Cyclin D1 mRNA expression after global DNA demethylation of breast cancer cell lines as determined by realtime PCR. Untreated tumor cells (black bars) and cells treated with DAC/TSA (grey bars) show significantly different expression levels of Cyclin D1 mRNA ( P = 0.029, two-sided Mann-Whitney U-test). Expression level of each sample is normalized to its GAPDH expression and related to untreated BT20 cells (set to 1).
    Figure Legend Snippet: Global demethylation and histone acetylation restores SFRP2 expression . (A) MSP of four malignant cell lines was performed with DNA from either untreated cells, or after treatment with 1 μM DAC, or after treatment with 300 nM TSA, or after a combined treatment applying both drugs. In three cell lines (BT20, MCF7, T47D) a promoter demethylating effect could be visually detected, since signals indicative of unmethylated SFRP2 promoter arise (BT20, MCF7) or become enhanced (T47D) after the combined treatment. In T47D, DAC alone had no detectable demethylating effect on the SFRP2 promoter. (B) Expression of SFRP2 mRNA before treatment, or after treatment with 1 μM DAC, or after treatment with 300 nM TSA, or after a combined treatment applying both drugs. Treatment with DAC alone was not able to induce SFRP2 expression in all cell lines, in contrast to TSA which induced expression in two out of four cell lines (SKBR3 and T47D) previously showing partial SFRP2 methylation. However, only combined promoter demethylation and histone reacetylation leads to strong induction of SFRP2 mRNA expression in all cell lines. GAPDH served as cDNA loading control. (C) Suppression of Cyclin D1 mRNA expression after global DNA demethylation of breast cancer cell lines as determined by realtime PCR. Untreated tumor cells (black bars) and cells treated with DAC/TSA (grey bars) show significantly different expression levels of Cyclin D1 mRNA ( P = 0.029, two-sided Mann-Whitney U-test). Expression level of each sample is normalized to its GAPDH expression and related to untreated BT20 cells (set to 1).

    Techniques Used: Expressing, Methylation, Polymerase Chain Reaction, MANN-WHITNEY

    SFRP2 methylation analysis of primary breast cancer specimens . MSP was performed on bisulfite-treated DNA from primary invasive breast cancer tissues. MSP results from 19 representative patient samples are shown. DNA bands in lanes labeled with U indicate PCR products amplified with primers recognizing the unmethylated SFRP2 promoter sequence. DNA bands in lanes labeled with M represent amplified products with methylation-specific primers. In addition, five representative normal cancer-unrelated breast tissues (N1 – N5) are shown. DNA from the breast cancer cell line BT20 and lymphocyte DNA from a healthy donor (Lyc) served as positive controls for MSP. NTC designates the no template control; T indicates tumor tissue; N indicates normal breast tissue.
    Figure Legend Snippet: SFRP2 methylation analysis of primary breast cancer specimens . MSP was performed on bisulfite-treated DNA from primary invasive breast cancer tissues. MSP results from 19 representative patient samples are shown. DNA bands in lanes labeled with U indicate PCR products amplified with primers recognizing the unmethylated SFRP2 promoter sequence. DNA bands in lanes labeled with M represent amplified products with methylation-specific primers. In addition, five representative normal cancer-unrelated breast tissues (N1 – N5) are shown. DNA from the breast cancer cell line BT20 and lymphocyte DNA from a healthy donor (Lyc) served as positive controls for MSP. NTC designates the no template control; T indicates tumor tissue; N indicates normal breast tissue.

    Techniques Used: Methylation, Labeling, Polymerase Chain Reaction, Amplification, Sequencing

    SFRP2 expression and promoter methylation in breast cancer cell lines . (A) SFRP2 mRNA expression in benign and malignant cell lines was determined by RT-PCR. All but one malignant cell line (Hs578T) completely lacked SFRP2 mRNA expression. Of the two benign breast cell lines (MCF10A and HMEC) only HMEC cells were found to express abundant SFRP2 mRNA. In a commercially available human normal breast tissue cDNA SFRP2 expression was clearly detectable, while expression was strongly reduced in a corresponding cDNA of human malignant breast tissue. (B) Genomic structure of the human SFRP2 gene on chromosome 4q31. Bioinformatic analysis revealed three CGIs (light blue), located within the SFRP2 promoter (left CGI), the 5' untranslated region (UTR; central CGI) and the coding sequence (CDS; right CGI). Methylation of the central CGI was explored by MSP. Circles indicate CpG sites; filled circles represent MSP forward (MSP-F) and reverse (MSP-R) primer hybridization sites. Indicated positions are related to the transcription start site (+1) initiating the 5'-UTR. (C) MSP was performed with bisulfite-treated DNA from the same breast cell lines as in A. DNA bands in lanes labeled with U indicate PCR products amplified with primers recognizing unmethylated SFRP2 promoter sequence. DNA bands in lanes labeled with M represent amplification products with methylation-specific primers. Five out of eight mammary tumor cell lines exhibit complete promoter methylation (MDA-MB-231, MCF7, MDA-MB-453, BT20 and BT474), two cell lines show partial SFRP2 methylation (SKBR3 and T47D). In Hs578T, only unmethylated SFRP2 promoter sequence could be detected, like it was also found in benign HMEC cells. In addition, lymphocyte DNA from a healthy donor did not reveal SFRP2 methylation. GAPDH served as cDNA loading control; NTC represents the no template control.
    Figure Legend Snippet: SFRP2 expression and promoter methylation in breast cancer cell lines . (A) SFRP2 mRNA expression in benign and malignant cell lines was determined by RT-PCR. All but one malignant cell line (Hs578T) completely lacked SFRP2 mRNA expression. Of the two benign breast cell lines (MCF10A and HMEC) only HMEC cells were found to express abundant SFRP2 mRNA. In a commercially available human normal breast tissue cDNA SFRP2 expression was clearly detectable, while expression was strongly reduced in a corresponding cDNA of human malignant breast tissue. (B) Genomic structure of the human SFRP2 gene on chromosome 4q31. Bioinformatic analysis revealed three CGIs (light blue), located within the SFRP2 promoter (left CGI), the 5' untranslated region (UTR; central CGI) and the coding sequence (CDS; right CGI). Methylation of the central CGI was explored by MSP. Circles indicate CpG sites; filled circles represent MSP forward (MSP-F) and reverse (MSP-R) primer hybridization sites. Indicated positions are related to the transcription start site (+1) initiating the 5'-UTR. (C) MSP was performed with bisulfite-treated DNA from the same breast cell lines as in A. DNA bands in lanes labeled with U indicate PCR products amplified with primers recognizing unmethylated SFRP2 promoter sequence. DNA bands in lanes labeled with M represent amplification products with methylation-specific primers. Five out of eight mammary tumor cell lines exhibit complete promoter methylation (MDA-MB-231, MCF7, MDA-MB-453, BT20 and BT474), two cell lines show partial SFRP2 methylation (SKBR3 and T47D). In Hs578T, only unmethylated SFRP2 promoter sequence could be detected, like it was also found in benign HMEC cells. In addition, lymphocyte DNA from a healthy donor did not reveal SFRP2 methylation. GAPDH served as cDNA loading control; NTC represents the no template control.

    Techniques Used: Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Hybridization, Labeling, Polymerase Chain Reaction, Amplification, Multiple Displacement Amplification

    46) Product Images from "Allele-specific transcriptional elongation regulates monoallelic expression of the IGF2BP1 gene"

    Article Title: Allele-specific transcriptional elongation regulates monoallelic expression of the IGF2BP1 gene

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-4-14

    DNA methylation analysis of the IGF2BP1 CTCF binding region . Analysis of DNA methylation with bisulfite sequencing at the intronic CTCF binding region of the IGF2BP1 gene is shown. (A) The percentage of methylation of CpG sites in gDNA derived from cell lines that express IGF2BP1 from only one allele (GM7016, GM6989) or from both alleles (GM7057) is shown. The CpG residue located within the CTCF binding motif is invariably methylated and is indicated by the thick black bar located adjacent to CpG site 7 (indicated by asterisks). (B) The percentage of methylation at each CpG site of the IGF2BP1 CTCF site in DNA samples recovered from anti-H3K9me3 ChIP. (C) The percentage of methylation at each CpG site of the IGF2BP1 CTCF site in DNA samples recovered from anti-CTCF ChIP experiments. The level of DNA methylation is represented according to the heat map keys located at the bottom of the figure.
    Figure Legend Snippet: DNA methylation analysis of the IGF2BP1 CTCF binding region . Analysis of DNA methylation with bisulfite sequencing at the intronic CTCF binding region of the IGF2BP1 gene is shown. (A) The percentage of methylation of CpG sites in gDNA derived from cell lines that express IGF2BP1 from only one allele (GM7016, GM6989) or from both alleles (GM7057) is shown. The CpG residue located within the CTCF binding motif is invariably methylated and is indicated by the thick black bar located adjacent to CpG site 7 (indicated by asterisks). (B) The percentage of methylation at each CpG site of the IGF2BP1 CTCF site in DNA samples recovered from anti-H3K9me3 ChIP. (C) The percentage of methylation at each CpG site of the IGF2BP1 CTCF site in DNA samples recovered from anti-CTCF ChIP experiments. The level of DNA methylation is represented according to the heat map keys located at the bottom of the figure.

    Techniques Used: DNA Methylation Assay, Binding Assay, Methylation Sequencing, Methylation, Derivative Assay, Chromatin Immunoprecipitation

    RNA polymerase II associates with both alleles in a monoallelically expressing cell line . (A) Recruitment of RNA polymerase II to the IGF2BP1 promoter was examined by ChIP in monoallelically expressing GM7007 cells. DNA recovered from chromatin that had been immunoprecipitated with anti-RNA polymerase II antibodies (Pol2) was amplified and sequenced for allelic association. Sequencing results (bottom) reveal that both alleles of the monoallelically expressing cell line GM7007 associate with RNA polymerase II near SNP site rs4794017. In contrast, sequencing of DNA from
    Figure Legend Snippet: RNA polymerase II associates with both alleles in a monoallelically expressing cell line . (A) Recruitment of RNA polymerase II to the IGF2BP1 promoter was examined by ChIP in monoallelically expressing GM7007 cells. DNA recovered from chromatin that had been immunoprecipitated with anti-RNA polymerase II antibodies (Pol2) was amplified and sequenced for allelic association. Sequencing results (bottom) reveal that both alleles of the monoallelically expressing cell line GM7007 associate with RNA polymerase II near SNP site rs4794017. In contrast, sequencing of DNA from "no antibody" ChIP reactions failed to produce sequence reads. (B) Allele specificity of precursor mRNA was determined by sequencing of cDNA prepared from total RNA of GM7007 cells. RNA had been extensively pretreated with DNase I to eliminate gDNA prior to reverse transcription by RT. Subsequently, cDNA samples were amplified using primers flanking rs4794017. In the absence of RT (-RT), no amplification products were oberved. +RT amplicons were gel-purified and sequenced. Bottom: Sequence traces at the heterozygous SNP site rs4794017 located 1 kb downstream of the transcription initiation site in cDNA of GM7007 indicate a single allele.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Sequencing, Purification

    Analysis of allele-specific IGF2BP1 expression . Comparative analysis of sequence variations in B lymphoblasts of the CEPH pedigree family 1331 reveals monoallelic expression of the IGF2BP1 gene. (A) Pedigree analysis was carried out for the SNP site rs11655950 located in the 3'-UTR of the IGF2BP1 gene. Each individual is shown with CEPH family identification, sample identification and genetic information (SNP genomic DNA (gDNA) genotype- or transcript-derived genotype). Individuals with monoallelic IGF2BP1 gene expression are indicated by asterisks. If the individual is homozygous at the SNP, allele-specific expression cannot be defined. (B) Left: Genotyping results at rs11655950 with gDNA from members of CEPH family 1331. gDNA was analyzed using the TaqMan SNP Genotyping Assay. This assay discriminates between sequence variants using two allele-specific probes carrying two different fluorophores, VIC and FAM. Individuals coded in red and green represent cell lines that are homozygous for alleles A and G, respectively. Orange-labeled individuals contain both A and G alleles at SNP rs11655950 and represent informative cell lines used for further analysis of monoallelic expression. Diamonds indicate cDNA samples, and black × indicates averaged triplicates of a no-template control (NTC) near the origin of the graph. Right: Genotyping results of transcript-derived cDNA from heterozygous B lymphoblasts. Individuals are color-coded in the figure key. No-RT controls (No RT) from cDNA synthesis are shown near the origin of the graph and are indicated by a black X. Control samples (standards) of stem cell lines previously genotyped as homozygous AA, heterozygous AG and homozygous GG were plotted and are indicated by diamonds.
    Figure Legend Snippet: Analysis of allele-specific IGF2BP1 expression . Comparative analysis of sequence variations in B lymphoblasts of the CEPH pedigree family 1331 reveals monoallelic expression of the IGF2BP1 gene. (A) Pedigree analysis was carried out for the SNP site rs11655950 located in the 3'-UTR of the IGF2BP1 gene. Each individual is shown with CEPH family identification, sample identification and genetic information (SNP genomic DNA (gDNA) genotype- or transcript-derived genotype). Individuals with monoallelic IGF2BP1 gene expression are indicated by asterisks. If the individual is homozygous at the SNP, allele-specific expression cannot be defined. (B) Left: Genotyping results at rs11655950 with gDNA from members of CEPH family 1331. gDNA was analyzed using the TaqMan SNP Genotyping Assay. This assay discriminates between sequence variants using two allele-specific probes carrying two different fluorophores, VIC and FAM. Individuals coded in red and green represent cell lines that are homozygous for alleles A and G, respectively. Orange-labeled individuals contain both A and G alleles at SNP rs11655950 and represent informative cell lines used for further analysis of monoallelic expression. Diamonds indicate cDNA samples, and black × indicates averaged triplicates of a no-template control (NTC) near the origin of the graph. Right: Genotyping results of transcript-derived cDNA from heterozygous B lymphoblasts. Individuals are color-coded in the figure key. No-RT controls (No RT) from cDNA synthesis are shown near the origin of the graph and are indicated by a black X. Control samples (standards) of stem cell lines previously genotyped as homozygous AA, heterozygous AG and homozygous GG were plotted and are indicated by diamonds.

    Techniques Used: Expressing, Sequencing, Derivative Assay, TaqMan SNP Genotyping Assay, Labeling

    Allelic specificity of CTCF and H3K9me3 . Informative ChIP templates were analyzed using the TaqMan allelic discrimination assay to address the allelic association of CTCF and H3K9me3. (A) Genotyping results at rs11870560 identify informative cell lines useful for the detection of allele-specific association of CTCF and H3K9me3. gDNA obtained from monoallelic and biallelic cell lines were genotyped using the TaqMan allelic discrimination assay. Squares represent gDNA samples and are coded in red and green to represent cell lines that are homozygous for allele C and allele T, respectively. Orange indicates heterozygous individuals. Averaged triplicate of a no-template control (NTC) is shown near the origin of the graph. (B) Genotyping at SNP rs11870560 with DNA templates recovered from ChIP experiments was used to identify the enrichment of the two alleles with either CTCF (circle) or H3K9me3 (triangle). Each color shown in the figure key represents a lymphoblastoid cell line (LCL) derived from an individual of the pedigree, while the shape represents the source of each sample (for example, squares signify input samples, while circles and triangles indicate ChIP samples obtained with CTCF and H3K9me3 antibodies, respectively). Immunoprecipitated templates were generated using the ChIP protocol described in Materials and Methods. Both monoallelic and biallelic cell lines indicate biallelic distribution of both CTCF and H3K9me3. Diamonds indicate control LCL samples (standards) previously genotyped as homozygous CC, heterozygous CT and homozygous TT.
    Figure Legend Snippet: Allelic specificity of CTCF and H3K9me3 . Informative ChIP templates were analyzed using the TaqMan allelic discrimination assay to address the allelic association of CTCF and H3K9me3. (A) Genotyping results at rs11870560 identify informative cell lines useful for the detection of allele-specific association of CTCF and H3K9me3. gDNA obtained from monoallelic and biallelic cell lines were genotyped using the TaqMan allelic discrimination assay. Squares represent gDNA samples and are coded in red and green to represent cell lines that are homozygous for allele C and allele T, respectively. Orange indicates heterozygous individuals. Averaged triplicate of a no-template control (NTC) is shown near the origin of the graph. (B) Genotyping at SNP rs11870560 with DNA templates recovered from ChIP experiments was used to identify the enrichment of the two alleles with either CTCF (circle) or H3K9me3 (triangle). Each color shown in the figure key represents a lymphoblastoid cell line (LCL) derived from an individual of the pedigree, while the shape represents the source of each sample (for example, squares signify input samples, while circles and triangles indicate ChIP samples obtained with CTCF and H3K9me3 antibodies, respectively). Immunoprecipitated templates were generated using the ChIP protocol described in Materials and Methods. Both monoallelic and biallelic cell lines indicate biallelic distribution of both CTCF and H3K9me3. Diamonds indicate control LCL samples (standards) previously genotyped as homozygous CC, heterozygous CT and homozygous TT.

    Techniques Used: Chromatin Immunoprecipitation, TaqMan Allelic Discrimination Assay, Derivative Assay, Immunoprecipitation, Generated

    47) Product Images from "Oestrogen receptor β regulates epigenetic patterns at specific genomic loci through interaction with thymine DNA glycosylase"

    Article Title: Oestrogen receptor β regulates epigenetic patterns at specific genomic loci through interaction with thymine DNA glycosylase

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-016-0055-7

    Hyper- but not hypomethylation in βerko MEFs is reversible by re-introduction of ERβ into βerko MEFs. a DNA methylation analysis of ten hypo- and eight hypermethylated positions. DNA methylation was assessed by methylation-specific enzymatic digest followed by qPCR. Positions with gene names in brackets were chosen for further analysis. b DNA methylation ( left panel ) and histone modifications ( right panel ) of differentially methylated genes in wt, βerko, and βerkohERβ MEFs. DNA methylation was assessed by pyrosequencing of bisulfite-treated DNA; black arrows mark DMPs identified by RRBS. Triple Asterisk indicates significant differences ( p
    Figure Legend Snippet: Hyper- but not hypomethylation in βerko MEFs is reversible by re-introduction of ERβ into βerko MEFs. a DNA methylation analysis of ten hypo- and eight hypermethylated positions. DNA methylation was assessed by methylation-specific enzymatic digest followed by qPCR. Positions with gene names in brackets were chosen for further analysis. b DNA methylation ( left panel ) and histone modifications ( right panel ) of differentially methylated genes in wt, βerko, and βerkohERβ MEFs. DNA methylation was assessed by pyrosequencing of bisulfite-treated DNA; black arrows mark DMPs identified by RRBS. Triple Asterisk indicates significant differences ( p

    Techniques Used: DNA Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    48) Product Images from "Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting"

    Article Title: Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005644

    Description of NLRP7 mutations with methylation and expression profiling of imprinted loci. (A) Confirmation of recessive NLRP7 mutations in female patients and heterozygous status in the RHM samples. The asterisk (*) on the electropherogram highlights the position of the mutation. For patient 3 the position of the deletion is shown. (B) Circular heat map of the 616 Infinium array probes mapping to 36 ubiquitously imprinted DMRs. The inner circle represents the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect NLRP7 mutations. (C) Confirmation of the methylation profile of the NLRP7 mutated RHMs at the NAP1L5 , PEG10 , RB1 , L3MBTL1 and H19 DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (D) Allelic expression analysis of imprinted genes NAP1L5 , HYMAI , PEG10 and PEG3 in control placenta samples (PL) and NLRP7 -mutated moles (RHM).
    Figure Legend Snippet: Description of NLRP7 mutations with methylation and expression profiling of imprinted loci. (A) Confirmation of recessive NLRP7 mutations in female patients and heterozygous status in the RHM samples. The asterisk (*) on the electropherogram highlights the position of the mutation. For patient 3 the position of the deletion is shown. (B) Circular heat map of the 616 Infinium array probes mapping to 36 ubiquitously imprinted DMRs. The inner circle represents the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect NLRP7 mutations. (C) Confirmation of the methylation profile of the NLRP7 mutated RHMs at the NAP1L5 , PEG10 , RB1 , L3MBTL1 and H19 DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (D) Allelic expression analysis of imprinted genes NAP1L5 , HYMAI , PEG10 and PEG3 in control placenta samples (PL) and NLRP7 -mutated moles (RHM).

    Techniques Used: Methylation, Expressing, Mutagenesis, Polymerase Chain Reaction, Subcloning, Amplification

    Methylation and expression analyses of placenta-specific DMRs in RHM samples. (A) Circular heatmap of the 153 Infinium array probes mapping to the 18 known placenta-specific imprinted DMRs. The inner circles represent the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect NLRP7 mutations. (B) Confirmation of the methylation profile at the maternally methylated GLIS3 , DNMT1 and MCCC1 DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (C) Allelic expression analysis of imprinted genes MCCC1 , LIN28B and GLIS3 in control placenta samples (PL) and NLRP7 -mutated moles (RHM). (D) Quantitative RT-PCR for H19 , DNMT1 and AGBL3 in RHM samples. The boxplot show the median expression (whiskers 5–95% percentile) determined for 15 control placenta samples with the values of RHMs highlighted.
    Figure Legend Snippet: Methylation and expression analyses of placenta-specific DMRs in RHM samples. (A) Circular heatmap of the 153 Infinium array probes mapping to the 18 known placenta-specific imprinted DMRs. The inner circles represent the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect NLRP7 mutations. (B) Confirmation of the methylation profile at the maternally methylated GLIS3 , DNMT1 and MCCC1 DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (C) Allelic expression analysis of imprinted genes MCCC1 , LIN28B and GLIS3 in control placenta samples (PL) and NLRP7 -mutated moles (RHM). (D) Quantitative RT-PCR for H19 , DNMT1 and AGBL3 in RHM samples. The boxplot show the median expression (whiskers 5–95% percentile) determined for 15 control placenta samples with the values of RHMs highlighted.

    Techniques Used: Methylation, Expressing, Polymerase Chain Reaction, Subcloning, Amplification, Quantitative RT-PCR

    Identification of additional placenta-specific imprinted DMRs in RHM samples. (A) A heatmap for the β mean of the Infinium probes with a methylation difference ( > 20%, minimum 3 consecutive probes) in RHMs associated with maternal effect NLRP7 mutations compared to control placental biopsies. (B) Schematic representation of the methylation-sensitive Hpa II genotyping assay. (C) Methylation profiles as determined by methylation-sensitive genotyping and (D) bisulfite PCR and subcloning on placenta and somatic tissue DNA samples at the SCIN , ST8AIA1 and CABIN1 promoters. Note that the samples used for methylation-sensitive genotyping and bisulphite PCR maybe different to highlight that methylation is not associated with genotype but parental origin.
    Figure Legend Snippet: Identification of additional placenta-specific imprinted DMRs in RHM samples. (A) A heatmap for the β mean of the Infinium probes with a methylation difference ( > 20%, minimum 3 consecutive probes) in RHMs associated with maternal effect NLRP7 mutations compared to control placental biopsies. (B) Schematic representation of the methylation-sensitive Hpa II genotyping assay. (C) Methylation profiles as determined by methylation-sensitive genotyping and (D) bisulfite PCR and subcloning on placenta and somatic tissue DNA samples at the SCIN , ST8AIA1 and CABIN1 promoters. Note that the samples used for methylation-sensitive genotyping and bisulphite PCR maybe different to highlight that methylation is not associated with genotype but parental origin.

    Techniques Used: Methylation, Genotyping Assay, Polymerase Chain Reaction, Subcloning

    49) Product Images from "ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma"

    Article Title: ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21149

    ARHI retained a hypermethylation copy in sporadic PCC (A) Schematic representations for the localization of CpG islands. Bottom boxes from left to right indicate CpG islands I, II, and III, respectively, and the red bar in shows the CpG sites. The corresponding numbers indicated the location in the genome from the UCSC database. (B) The ARHI promoter included three CpG islands; the methylation ratios were detected by EpiTYPER methylation analysis. The methylation ratios were significantly higher in PCC tumors than normal adrenal tissues. The methylation ratios of the three CpG islands were obviously higher in the tumors with deleted copy numbers of ARHI when compared with those that carried normal copy numbers of ARHI. (C) Correlation between ARHI promoter CpG islands and their expression in PCC samples (n=38). (D) Summary of bisulfite-treated genomic DNA sequencing of PCC samples dependent on ARHI deletion, where the amplified region includes all three CpG islands; 73 CpG dinucleotides (CpGs), represented by circles located on the region, were analyzed by DNA sequencing. Black and white circles represent the methylated and unmethylated CpG dinucleotides, respectively. Each line represents the DNA sequence of a random clone, of which black and white circles represent unmethylated and methylated CpG sites of these regions, respectively. (E) Fluorescence in situ hybridization studies in Subjects 1 and 2. In Subjects 1, the 1chr.p31.3 (ARHI) labeled with Rhodamine BAC clone showed 2 copies (panel a) while the “control”chr.1q21 labeled with FITC BAC clone showed normal hybridization pattern in nuclei (Green). Subjects 2 showed the ARHI deletion (panel b) detected by the Rhodamine BAC clone (arrow). The FITC BAC clone was the control probe. (F) PHPC with ARHI copy number deletion (without endogenous ARHI expression) and with normal ARHI copy number were used to detect the methylation status using bisulfite-treated genomic DNA sequencing. Western blot and RT-PCR were used to determine whether ARHI was expressed at protein and mRNA levels. (G) Effect of DAC expression on methylation status of the ARHI gene promoter. DNA from control or DAC-treated PHPC with negative ARHI expression were collected at the indicated time points, cloned and sequenced to detect CpG-island methylation of the ARHI promoter. (H) ARHI-negative PHPC were treated with DAC, after 24h, 48h, 72h; ARHI was detected using RT-PCR and western blot, the error bars are represented as mean ± SD. (I) ARHI methylated allele analysis and its maternal imprinting in PCC tumors. SNP rs11209207 of normal DNA from 4 PCC patients (lane 1:N1-N4): one allele is lost in tumor DNA (lane 4:P1-P4); the retained allele is methylated (lane 5:Methylated allele1-4). Genotype of SNP rs11209207 of 4 families was shown. Maternal (lane 2:M1-M4); paternal (lane 3:F1-F4).
    Figure Legend Snippet: ARHI retained a hypermethylation copy in sporadic PCC (A) Schematic representations for the localization of CpG islands. Bottom boxes from left to right indicate CpG islands I, II, and III, respectively, and the red bar in shows the CpG sites. The corresponding numbers indicated the location in the genome from the UCSC database. (B) The ARHI promoter included three CpG islands; the methylation ratios were detected by EpiTYPER methylation analysis. The methylation ratios were significantly higher in PCC tumors than normal adrenal tissues. The methylation ratios of the three CpG islands were obviously higher in the tumors with deleted copy numbers of ARHI when compared with those that carried normal copy numbers of ARHI. (C) Correlation between ARHI promoter CpG islands and their expression in PCC samples (n=38). (D) Summary of bisulfite-treated genomic DNA sequencing of PCC samples dependent on ARHI deletion, where the amplified region includes all three CpG islands; 73 CpG dinucleotides (CpGs), represented by circles located on the region, were analyzed by DNA sequencing. Black and white circles represent the methylated and unmethylated CpG dinucleotides, respectively. Each line represents the DNA sequence of a random clone, of which black and white circles represent unmethylated and methylated CpG sites of these regions, respectively. (E) Fluorescence in situ hybridization studies in Subjects 1 and 2. In Subjects 1, the 1chr.p31.3 (ARHI) labeled with Rhodamine BAC clone showed 2 copies (panel a) while the “control”chr.1q21 labeled with FITC BAC clone showed normal hybridization pattern in nuclei (Green). Subjects 2 showed the ARHI deletion (panel b) detected by the Rhodamine BAC clone (arrow). The FITC BAC clone was the control probe. (F) PHPC with ARHI copy number deletion (without endogenous ARHI expression) and with normal ARHI copy number were used to detect the methylation status using bisulfite-treated genomic DNA sequencing. Western blot and RT-PCR were used to determine whether ARHI was expressed at protein and mRNA levels. (G) Effect of DAC expression on methylation status of the ARHI gene promoter. DNA from control or DAC-treated PHPC with negative ARHI expression were collected at the indicated time points, cloned and sequenced to detect CpG-island methylation of the ARHI promoter. (H) ARHI-negative PHPC were treated with DAC, after 24h, 48h, 72h; ARHI was detected using RT-PCR and western blot, the error bars are represented as mean ± SD. (I) ARHI methylated allele analysis and its maternal imprinting in PCC tumors. SNP rs11209207 of normal DNA from 4 PCC patients (lane 1:N1-N4): one allele is lost in tumor DNA (lane 4:P1-P4); the retained allele is methylated (lane 5:Methylated allele1-4). Genotype of SNP rs11209207 of 4 families was shown. Maternal (lane 2:M1-M4); paternal (lane 3:F1-F4).

    Techniques Used: Periodic Counter-current Chromatography, Methylation, Expressing, DNA Sequencing, Amplification, Sequencing, Fluorescence, In Situ Hybridization, Labeling, BAC Assay, Hybridization, Western Blot, Reverse Transcription Polymerase Chain Reaction, Clone Assay

    50) Product Images from "Regions of common inter-individual DNA methylation differences in human monocytes: genetic basis and potential function"

    Article Title: Regions of common inter-individual DNA methylation differences in human monocytes: genetic basis and potential function

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-017-0144-2

    Correlation between DNA methylation and gene expression. a Scatter plot of the differences in gene expression levels of the putative target genes identified by GREAT and the differences in DMR methylation in donors Hm03 and Hm05. b Scatter plot of the differences in transcript isoform levels of genes harboring a DMR and the differences in methylation of the 77 intragenic DMRs
    Figure Legend Snippet: Correlation between DNA methylation and gene expression. a Scatter plot of the differences in gene expression levels of the putative target genes identified by GREAT and the differences in DMR methylation in donors Hm03 and Hm05. b Scatter plot of the differences in transcript isoform levels of genes harboring a DMR and the differences in methylation of the 77 intragenic DMRs

    Techniques Used: DNA Methylation Assay, Expressing, Methylation

    Histone modifications of 2 kb regions centered on the 157 inter-individual DMRs. a Heatmaps of histone modification signals for Hm03 ( left ) and Hm05 ( right ). Heatmaps show log2 ratio ChIP signal over input for six different histone modifications. b Scatter plots showing difference in histone modification signals between Hm05 and Hm03 as a function of methylation differences between the two donors. Active histone marks are inversely correlated with DNA methylation (linear regression)
    Figure Legend Snippet: Histone modifications of 2 kb regions centered on the 157 inter-individual DMRs. a Heatmaps of histone modification signals for Hm03 ( left ) and Hm05 ( right ). Heatmaps show log2 ratio ChIP signal over input for six different histone modifications. b Scatter plots showing difference in histone modification signals between Hm05 and Hm03 as a function of methylation differences between the two donors. Active histone marks are inversely correlated with DNA methylation (linear regression)

    Techniques Used: Modification, Chromatin Immunoprecipitation, Methylation, DNA Methylation Assay

    51) Product Images from "Methylation of mitochondrial DNA displacement loop region regulates mitochondrial copy number in colorectal cancer"

    Article Title: Methylation of mitochondrial DNA displacement loop region regulates mitochondrial copy number in colorectal cancer

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7264

    Target mtDNA D-loop promoter and mtDNA methylation analysis. (A) Target promoter with 14 CpG sites (divided into 12 CpG units) in base pairs 302 to 766 of the D-loop promoter. Note, the 2nd, 10, 16, and 18th CpG sites were not available to be examined. (B) mtDNA methylation analysis by Sequenom MassARRAY platform, showing methylation levels of CpG sites for each cell line with or without 5 µM 5-AZA treatment. D-loop, displacement loop; DMSO, dimethyl sulfoxide; 5-AZA, 5-aza-2′-deoxycytidine; mtDNA, mitochondrial DNA.
    Figure Legend Snippet: Target mtDNA D-loop promoter and mtDNA methylation analysis. (A) Target promoter with 14 CpG sites (divided into 12 CpG units) in base pairs 302 to 766 of the D-loop promoter. Note, the 2nd, 10, 16, and 18th CpG sites were not available to be examined. (B) mtDNA methylation analysis by Sequenom MassARRAY platform, showing methylation levels of CpG sites for each cell line with or without 5 µM 5-AZA treatment. D-loop, displacement loop; DMSO, dimethyl sulfoxide; 5-AZA, 5-aza-2′-deoxycytidine; mtDNA, mitochondrial DNA.

    Techniques Used: Methylation

    52) Product Images from "Differences in DNA Methylation and Functional Expression in Lactase Persistent and Non-persistent Individuals"

    Article Title: Differences in DNA Methylation and Functional Expression in Lactase Persistent and Non-persistent Individuals

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23957-4

    Pyrosequencing of DNA methylation at the LCT enhancer. ( A ) Schematic of LCT enhancer with location of the three CpGs under investigation. The position of −13910C > T (rs4988235) in relation to the CpGs and the genomic coordinates of the PCR product are also given. ( B) DNA methylation quantification in CC vs. C/T vs. TT individuals, as well as in ( C) Phenotypically Persistent (PER) and Non-persistent (NON-PER) individuals. Student’s T-test was calculated for inter-group comparisons (*p
    Figure Legend Snippet: Pyrosequencing of DNA methylation at the LCT enhancer. ( A ) Schematic of LCT enhancer with location of the three CpGs under investigation. The position of −13910C > T (rs4988235) in relation to the CpGs and the genomic coordinates of the PCR product are also given. ( B) DNA methylation quantification in CC vs. C/T vs. TT individuals, as well as in ( C) Phenotypically Persistent (PER) and Non-persistent (NON-PER) individuals. Student’s T-test was calculated for inter-group comparisons (*p

    Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction

    Pyrosequencing of DNA methylation at cg20242066. ( A ) Schematic of LCT proximal promoter with location of cg20242066 upstream of the first three exons of the gene. The genomic coordinate of cg20242066 is also given (genome build hg19). ( B) DNA methylation quantification in CC vs. C/T vs. TT individuals, as well as in ( C) Phenotypically Persistent (PER) and Non-persistent (NON-PER) individuals. Student’s T-test was calculated for inter-group comparisons (*p
    Figure Legend Snippet: Pyrosequencing of DNA methylation at cg20242066. ( A ) Schematic of LCT proximal promoter with location of cg20242066 upstream of the first three exons of the gene. The genomic coordinate of cg20242066 is also given (genome build hg19). ( B) DNA methylation quantification in CC vs. C/T vs. TT individuals, as well as in ( C) Phenotypically Persistent (PER) and Non-persistent (NON-PER) individuals. Student’s T-test was calculated for inter-group comparisons (*p

    Techniques Used: DNA Methylation Assay

    53) Product Images from "Aberrant Promoter Methylation of YAP Gene and its Subsequent Downregulation in Indian Breast Cancer Patients"

    Article Title: Aberrant Promoter Methylation of YAP Gene and its Subsequent Downregulation in Indian Breast Cancer Patients

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4627-8

    Methylation-specific PCR analysis of YAP gene in breast cancer patients: L 1kb DNA ladder, M methylated YAP promoter (PCR product size-187 bp), UM unmethylated YAP promoter (PCR product size-188 bp), PC positive control for methylated and unmethylated alleles (Completely methylated and unmethylated DNA controls, respectively), N normal breast sample, and T breast tumor sample
    Figure Legend Snippet: Methylation-specific PCR analysis of YAP gene in breast cancer patients: L 1kb DNA ladder, M methylated YAP promoter (PCR product size-187 bp), UM unmethylated YAP promoter (PCR product size-188 bp), PC positive control for methylated and unmethylated alleles (Completely methylated and unmethylated DNA controls, respectively), N normal breast sample, and T breast tumor sample

    Techniques Used: Methylation, Polymerase Chain Reaction, Positive Control

    54) Product Images from "Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2"

    Article Title: Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00967

    Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.
    Figure Legend Snippet: Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.

    Techniques Used: Mutagenesis, Expressing, Polymerase Chain Reaction, Hybridization, CTG Assay, Labeling, Quantitative RT-PCR

    55) Product Images from "MicroRNA-335 inhibits tumor reinitiation and is silenced through genetic and epigenetic mechanisms in human breast cancer"

    Article Title: MicroRNA-335 inhibits tumor reinitiation and is silenced through genetic and epigenetic mechanisms in human breast cancer

    Journal: Genes & Development

    doi: 10.1101/gad.1974211

    Pyrosequencing reveals the methylation status of CpG island 3 as a predictor of miR-335 expression, and inhibition of DNA methylation is sufficient to restore miR-335 expression in metastatic breast cancer cells. ( A–D ) Pyrosequencing of bisulfite-treated DNA reveals the methylation percentage ( Y -axis) as a function of CpG dinucleotide position in island 3 of poorly metastatic breast cancer populations (MDA parental and CN34 parental) and their highly metastatic derivatives (LM2 and CN34LM1A). All error bars represent SEM. ( E ) CpG methylation percentage as a function of miR-335 expression (correlation coefficient r 2 = −0.81; P = 0.004). ( F ) Quantitative real-time PCR expression of miR-335 expression in parental MDA-231 breast cancer line and its metastatic LM2 derivative and the CN34 primary malignant population and its metastatic derivative (CNLM1A) in the presence or absence of 5-Aza (5 μM treatment for 96 h for MDA lines and 1 μM for CN34 lines; metastatic lines, n = 6; parental lines, n = 3). P -values represent unpaired one-tailed t -test significance values. (*) P
    Figure Legend Snippet: Pyrosequencing reveals the methylation status of CpG island 3 as a predictor of miR-335 expression, and inhibition of DNA methylation is sufficient to restore miR-335 expression in metastatic breast cancer cells. ( A–D ) Pyrosequencing of bisulfite-treated DNA reveals the methylation percentage ( Y -axis) as a function of CpG dinucleotide position in island 3 of poorly metastatic breast cancer populations (MDA parental and CN34 parental) and their highly metastatic derivatives (LM2 and CN34LM1A). All error bars represent SEM. ( E ) CpG methylation percentage as a function of miR-335 expression (correlation coefficient r 2 = −0.81; P = 0.004). ( F ) Quantitative real-time PCR expression of miR-335 expression in parental MDA-231 breast cancer line and its metastatic LM2 derivative and the CN34 primary malignant population and its metastatic derivative (CNLM1A) in the presence or absence of 5-Aza (5 μM treatment for 96 h for MDA lines and 1 μM for CN34 lines; metastatic lines, n = 6; parental lines, n = 3). P -values represent unpaired one-tailed t -test significance values. (*) P

    Techniques Used: Methylation, Expressing, Inhibition, DNA Methylation Assay, Multiple Displacement Amplification, CpG Methylation Assay, Real-time Polymerase Chain Reaction, One-tailed Test

    56) Product Images from "Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes"

    Article Title: Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1004487107

    Methylation imprint at the PWS-IC is maintained during reprogramming. ( A ) Methylation-specific PCR analysis of genomic DNA from normal, AS, and PWS fibroblasts. Primers specific for the methylated allele amplify a band that is 174 bp, whereas primers
    Figure Legend Snippet: Methylation imprint at the PWS-IC is maintained during reprogramming. ( A ) Methylation-specific PCR analysis of genomic DNA from normal, AS, and PWS fibroblasts. Primers specific for the methylated allele amplify a band that is 174 bp, whereas primers

    Techniques Used: Methylation, Polymerase Chain Reaction

    57) Product Images from "Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter"

    Article Title: Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1019

    ICAM-1 -targeted DNA demethylation. ( A ) Schematic representation of targeted DNA demethylation in ICAM-1 promoter by epigenetic editing. The binding sites of the ZF in the promoter of ICAM-1 are depicted, and a magnification of the target region and the actual position of each selected CpG from the transcription start site (target CpG sites are numbered #10–14, with #10 and #11 located within the ZF binding region). The purple area represents the ZFP binding site. Gray ovals represent the ZF modules, and the red ovals represent the epigenetic effector domain. A six ZFP is fused to candidate epigenetic effector domains or to the transcription activator VP64. The candidate effector domains are shown in the lower right portion of the panel: the transcription activator VP64 as the well-known positive control and the CDs of the mouse Ten-Eleven Translocation proteins (TET1, -2 and -3). Rectangular boxes display the functional domains as explained in the key box. ( B ) Protein expression of ZF fusion constructs in A2780 host cells. Upper panel: conventional western blot could only detect ZF-VP64 and ZF-only (ZF-noED); middle panel: HA-tag immunoprecipitation followed by western blot detected all ZF fusion constructs; lower panel: beta-actin was used as an input control. ( C ) Quantitative analysis of the methylation levels of target CpG sites in ZF binding region by pyrosequencing after treatment with the ICAM-1 -targeted candidate demethylation effector domains in unsorted and sorted A2780 ovarian cancer cells. ( D ) Quantitative analysis of the methylation levels of target CpG sites in effector domain-targeted region by pyrosequencing after treatment with the ICAM-1 -targeted candidate demethylation effector domains in unsorted and sorted A2780 ovarian cancer cells. ( E ) Examination of 5-hydroxymethylcytosine levels at ICAM-1 promoter target region in unsorted A2780 ovarian cancer cells transduced to express CD54-TET1 or CD54-TET2CD. Quantitative PCR was performed on A2780 genomic DNA immunoprecipitated using anti-5-mC antibody (MeDIP) or anti-5-hmC antibody (hMeDIP) to evaluate the relative 5-hmC and 5-mC levels (IP/input) at the ICAM-1 promoter. pMXempty serves as a negative control. ( F ) Quantitative sequencing analysis of methylation and hydroxymethylation levels of target CpG sites at single-base resolution by combining oxidative bisulfite treatment and pyrosequencing in sorted A2780 ovarian cancer cells transduced to express CD54-TET1 or CD54-TET2CD.
    Figure Legend Snippet: ICAM-1 -targeted DNA demethylation. ( A ) Schematic representation of targeted DNA demethylation in ICAM-1 promoter by epigenetic editing. The binding sites of the ZF in the promoter of ICAM-1 are depicted, and a magnification of the target region and the actual position of each selected CpG from the transcription start site (target CpG sites are numbered #10–14, with #10 and #11 located within the ZF binding region). The purple area represents the ZFP binding site. Gray ovals represent the ZF modules, and the red ovals represent the epigenetic effector domain. A six ZFP is fused to candidate epigenetic effector domains or to the transcription activator VP64. The candidate effector domains are shown in the lower right portion of the panel: the transcription activator VP64 as the well-known positive control and the CDs of the mouse Ten-Eleven Translocation proteins (TET1, -2 and -3). Rectangular boxes display the functional domains as explained in the key box. ( B ) Protein expression of ZF fusion constructs in A2780 host cells. Upper panel: conventional western blot could only detect ZF-VP64 and ZF-only (ZF-noED); middle panel: HA-tag immunoprecipitation followed by western blot detected all ZF fusion constructs; lower panel: beta-actin was used as an input control. ( C ) Quantitative analysis of the methylation levels of target CpG sites in ZF binding region by pyrosequencing after treatment with the ICAM-1 -targeted candidate demethylation effector domains in unsorted and sorted A2780 ovarian cancer cells. ( D ) Quantitative analysis of the methylation levels of target CpG sites in effector domain-targeted region by pyrosequencing after treatment with the ICAM-1 -targeted candidate demethylation effector domains in unsorted and sorted A2780 ovarian cancer cells. ( E ) Examination of 5-hydroxymethylcytosine levels at ICAM-1 promoter target region in unsorted A2780 ovarian cancer cells transduced to express CD54-TET1 or CD54-TET2CD. Quantitative PCR was performed on A2780 genomic DNA immunoprecipitated using anti-5-mC antibody (MeDIP) or anti-5-hmC antibody (hMeDIP) to evaluate the relative 5-hmC and 5-mC levels (IP/input) at the ICAM-1 promoter. pMXempty serves as a negative control. ( F ) Quantitative sequencing analysis of methylation and hydroxymethylation levels of target CpG sites at single-base resolution by combining oxidative bisulfite treatment and pyrosequencing in sorted A2780 ovarian cancer cells transduced to express CD54-TET1 or CD54-TET2CD.

    Techniques Used: Binding Assay, Positive Control, Translocation Assay, Functional Assay, Expressing, Construct, Western Blot, Immunoprecipitation, Methylation, Real-time Polymerase Chain Reaction, Methylated DNA Immunoprecipitation, Negative Control, Sequencing

    Genome-wide DNA demethylation effects by targeted TET-fusions. ( A ) Fusion of the TET1 and -2 CDs to the ICAM-1 -targeting DNA binding domains CD54 did result in genome-wide induction of hydroxymethylation. DNA dot-blot assays were performed with genomic DNA isolated from unsorted and sorted A2780 ovarian cancer cells transduced to express pMX-CD54-TET1 or -2CD. ( B ) 5-mC and 5-hmC levels at human long interspersed nuclear element-1 ( LINE-1 ) in HEK293T cells transfected with pcDNA-TET1 CD. Quantitative PCR was performed on genomic DNA immunoprecipitated using anti-5-mC antibody (for MeDIP) or anti-5-hmC antibody (for hMeDIP) to evaluate the relative 5-hmC and 5-mC levels (IP/input) at the LINE-1 . Genomic DNA from HEK293T cells transfected with pcDNAempty serves as a negative control ( C ) Quantitative analysis of the methylation levels of core CpG sites in LINE-1 promoter by pyrosequencing after treatment with the untargeted candidate demethylation effector domains TET1 and -2CD as well as catalytically inactive TET1 and -2CD mutant in A2780 ovarian cancer cells. ( D ) Quantitative analysis of the methylation levels of core CpG sites in LINE-1 promoter by pyrosequencing after treatment with the ICAM-1- and EpCAM- targeted candidate demethylation effector domains in unsorted and sorted A2780 ovarian cancer cells. The results are shown as the mean methylation of three CpG sites.
    Figure Legend Snippet: Genome-wide DNA demethylation effects by targeted TET-fusions. ( A ) Fusion of the TET1 and -2 CDs to the ICAM-1 -targeting DNA binding domains CD54 did result in genome-wide induction of hydroxymethylation. DNA dot-blot assays were performed with genomic DNA isolated from unsorted and sorted A2780 ovarian cancer cells transduced to express pMX-CD54-TET1 or -2CD. ( B ) 5-mC and 5-hmC levels at human long interspersed nuclear element-1 ( LINE-1 ) in HEK293T cells transfected with pcDNA-TET1 CD. Quantitative PCR was performed on genomic DNA immunoprecipitated using anti-5-mC antibody (for MeDIP) or anti-5-hmC antibody (for hMeDIP) to evaluate the relative 5-hmC and 5-mC levels (IP/input) at the LINE-1 . Genomic DNA from HEK293T cells transfected with pcDNAempty serves as a negative control ( C ) Quantitative analysis of the methylation levels of core CpG sites in LINE-1 promoter by pyrosequencing after treatment with the untargeted candidate demethylation effector domains TET1 and -2CD as well as catalytically inactive TET1 and -2CD mutant in A2780 ovarian cancer cells. ( D ) Quantitative analysis of the methylation levels of core CpG sites in LINE-1 promoter by pyrosequencing after treatment with the ICAM-1- and EpCAM- targeted candidate demethylation effector domains in unsorted and sorted A2780 ovarian cancer cells. The results are shown as the mean methylation of three CpG sites.

    Techniques Used: Genome Wide, Binding Assay, Dot Blot, Isolation, Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Methylated DNA Immunoprecipitation, Negative Control, Methylation, Mutagenesis

    58) Product Images from "Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice"

    Article Title: Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094735

    The DNA methylation status of Aid −/− iPS cells. (A–D) The DNA methylation status of the Nanog promoter (A), Oct3/4 promoter (B), B1 (C) and LINE1 (D) detected by pyrosequencing. The iPS cell clones analyzed were the same as those examined in Fig. 2C . The data are represented as the averages ± SD of the clones. (E) The results of the comprehensive DNA methylation analysis with MBD-sequencing. Pie charts show the comparison of the detected methylated regions between Aid +/+ iPS cells and Aid +/+ MEFs (left), Aid +/+ iPS cells and ES cells (middle), and Aid +/+ iPS cells and Aid −/− iPS cells (right). DMRs; Differentially methylated regions, CMRs; Commonly methylated regions.
    Figure Legend Snippet: The DNA methylation status of Aid −/− iPS cells. (A–D) The DNA methylation status of the Nanog promoter (A), Oct3/4 promoter (B), B1 (C) and LINE1 (D) detected by pyrosequencing. The iPS cell clones analyzed were the same as those examined in Fig. 2C . The data are represented as the averages ± SD of the clones. (E) The results of the comprehensive DNA methylation analysis with MBD-sequencing. Pie charts show the comparison of the detected methylated regions between Aid +/+ iPS cells and Aid +/+ MEFs (left), Aid +/+ iPS cells and ES cells (middle), and Aid +/+ iPS cells and Aid −/− iPS cells (right). DMRs; Differentially methylated regions, CMRs; Commonly methylated regions.

    Techniques Used: DNA Methylation Assay, Clone Assay, Sequencing, Methylation

    59) Product Images from "Targeted DNA Demethylation and Endogenous Gene Activation Using Programmable TALE-TET1 Fusions"

    Article Title: Targeted DNA Demethylation and Endogenous Gene Activation Using Programmable TALE-TET1 Fusions

    Journal: Nature biotechnology

    doi: 10.1038/nbt.2726

    Targeted demethylation and activation of the human RHOXF2 gene by TALE-TET1 Fusion Proteins (A) Schematic illustrating the human RHOXF2 locus with CpGs indicated with black arrows. Red arrows indicate the location and direction (5’ to 3’) of 11 TALE-TET1 fusion protein binding sites. Numbering indicates position on the DNA relative to the start site of transcription (right-angle arrow). (B) Expression levels of RHOXF2 mRNA (normalized to β-actin mRNA levels) in HeLa cells transfected with plasmids expressing RH-3 or RH-4 TALE-TET1 proteins, RH-3 or RH-4 TALE-TET1 proteins bearing mutations that inactivate TET1 catalytic function, or an off-target TALE-TET1 protein (targeted to KLF4 ) or a control GFP expression plasmid. Means of three independent samples each assayed three times by quantitative RT-PCR are shown with bars representing standard errors of the mean. Asterisks indicate samples with values significantly greater than those obtained with the off-target control as determined by a one-sided Welch’s t-test (n=3, p
    Figure Legend Snippet: Targeted demethylation and activation of the human RHOXF2 gene by TALE-TET1 Fusion Proteins (A) Schematic illustrating the human RHOXF2 locus with CpGs indicated with black arrows. Red arrows indicate the location and direction (5’ to 3’) of 11 TALE-TET1 fusion protein binding sites. Numbering indicates position on the DNA relative to the start site of transcription (right-angle arrow). (B) Expression levels of RHOXF2 mRNA (normalized to β-actin mRNA levels) in HeLa cells transfected with plasmids expressing RH-3 or RH-4 TALE-TET1 proteins, RH-3 or RH-4 TALE-TET1 proteins bearing mutations that inactivate TET1 catalytic function, or an off-target TALE-TET1 protein (targeted to KLF4 ) or a control GFP expression plasmid. Means of three independent samples each assayed three times by quantitative RT-PCR are shown with bars representing standard errors of the mean. Asterisks indicate samples with values significantly greater than those obtained with the off-target control as determined by a one-sided Welch’s t-test (n=3, p

    Techniques Used: Activation Assay, Protein Binding, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR

    Targeted demethylation and activation of the human HBB gene by TALE-TET1 Fusion Proteins (A) Schematic illustrating the human HBB locus with CpGs indicated with black arrows. Numbering indicates position on the DNA relative to the start site of transcription (right-angle arrow). Colored arrows indicate the location and direction (5’ to 3’) of 10 TALE-TET1 fusion protein binding sites. (B) Demethylation activities of HB-1, -2, -3, -4, -5, and -6 TALE-TET1 fusion proteins in K562 cells. Graphs show the fraction of CpGs methylated (y-axis) for different positions along the length of the HBB promoter (x-axis, numbered relative to the transcription start site) in K562 cells transfected with plasmids expressing one of the six HB TALE-TET1 fusion proteins (colored circles), an off-target TALE-TET1 fusion to a site in the KLF4 gene (black triangles), or GFP (green triangles). Each data point represents the mean of three independent transfection experiments with bars indicating standard errors of the mean. Methylation status for each experiment was assessed using high-throughput bisulfite sequencing. Note that the GFP and off-target TALE-TET1 control data points shown are the same in both panels and are depicted twice for ease of comparison with experimental samples. Colored arrows indicate the location and direction (5’ to 3’) of the various HB TALE-TET1 binding sites. (C) Demethylation activities of HB-7, -8, -9, and -10 TALE-TET1 fusion proteins in K562 cells. Data represented as in (B) (D) Expression levels of HBB mRNA (normalized to β-actin mRNA levels) in K562 cells transfected with indicated TALE-TET1 fusion protein expression plasmids or a control GFP expression plasmid. Means of three independent samples assayed by quantitative RT-PCR are shown with bars representing standard errors of the mean. Asterisks indicate samples with values significantly greater than those obtained with an off-target control TALE-TET1 fusion protein (targeted to a site in the human KLF4 gene) as determined by a one-sided Welch’s t-test (n=3, p
    Figure Legend Snippet: Targeted demethylation and activation of the human HBB gene by TALE-TET1 Fusion Proteins (A) Schematic illustrating the human HBB locus with CpGs indicated with black arrows. Numbering indicates position on the DNA relative to the start site of transcription (right-angle arrow). Colored arrows indicate the location and direction (5’ to 3’) of 10 TALE-TET1 fusion protein binding sites. (B) Demethylation activities of HB-1, -2, -3, -4, -5, and -6 TALE-TET1 fusion proteins in K562 cells. Graphs show the fraction of CpGs methylated (y-axis) for different positions along the length of the HBB promoter (x-axis, numbered relative to the transcription start site) in K562 cells transfected with plasmids expressing one of the six HB TALE-TET1 fusion proteins (colored circles), an off-target TALE-TET1 fusion to a site in the KLF4 gene (black triangles), or GFP (green triangles). Each data point represents the mean of three independent transfection experiments with bars indicating standard errors of the mean. Methylation status for each experiment was assessed using high-throughput bisulfite sequencing. Note that the GFP and off-target TALE-TET1 control data points shown are the same in both panels and are depicted twice for ease of comparison with experimental samples. Colored arrows indicate the location and direction (5’ to 3’) of the various HB TALE-TET1 binding sites. (C) Demethylation activities of HB-7, -8, -9, and -10 TALE-TET1 fusion proteins in K562 cells. Data represented as in (B) (D) Expression levels of HBB mRNA (normalized to β-actin mRNA levels) in K562 cells transfected with indicated TALE-TET1 fusion protein expression plasmids or a control GFP expression plasmid. Means of three independent samples assayed by quantitative RT-PCR are shown with bars representing standard errors of the mean. Asterisks indicate samples with values significantly greater than those obtained with an off-target control TALE-TET1 fusion protein (targeted to a site in the human KLF4 gene) as determined by a one-sided Welch’s t-test (n=3, p

    Techniques Used: Activation Assay, Protein Binding, Methylation, Transfection, Expressing, High Throughput Screening Assay, Methylation Sequencing, Binding Assay, Plasmid Preparation, Quantitative RT-PCR

    60) Product Images from "EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation"

    Article Title: EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation

    Journal: Genome Biology

    doi: 10.1186/s13059-019-1853-6

    EpiMethylTag is a reproducible method for testing whether DNAme can coexist with TF binding (CTCF) or chromatin accessibility genome-wide. a Pearson correlation of read counts comparing M-ATAC with unconverted samples (NC) and regular ATAC-seq (top), and CTCF M-ChIP with unconverted samples, a sample from the Schubeler lab generated using ChIP-BisSeq [ 1 ] (GSE39739) and regular CTCF ChIP-seq (bottom). b Representative IGV screenshots of EpiMethylTag, at the Klf4 locus (left panel), the Pisd-ps1 locus (middle panel), and the Slc5a8 locus (right panel). ATAC and M-ATAC in green, CTCF in purple and DNA methylation from merged M-ATAC, merged CTCF M-ChIP and WGBS (methylation from 0% in blue to 100% in red). A zoom-in of methylation at the highlighted region is shown at the bottom of each example. The Klf4 locus illustrates a region that has low methylation as detected by M-ATAC, CTCF M-ChIP, and WGBS. The Pisd-ps1 locus illustrates a region that has high methylation as detected by M-ATAC, CTCF M-ChIP, and WGBS. The Slc5a8 locus illustrates a region that has low methylation as detected by M-ATAC and high methylation as detected by WGBS. c Density plots of methylation from EpiMethyltag compared with WGBS. Only CpGs inside peaks and with at least five reads were considered. Top: average methylation of CpGs per M-ATAC peak in M-ATAC versus WGBS (Pearson correlation = 0.69, p value
    Figure Legend Snippet: EpiMethylTag is a reproducible method for testing whether DNAme can coexist with TF binding (CTCF) or chromatin accessibility genome-wide. a Pearson correlation of read counts comparing M-ATAC with unconverted samples (NC) and regular ATAC-seq (top), and CTCF M-ChIP with unconverted samples, a sample from the Schubeler lab generated using ChIP-BisSeq [ 1 ] (GSE39739) and regular CTCF ChIP-seq (bottom). b Representative IGV screenshots of EpiMethylTag, at the Klf4 locus (left panel), the Pisd-ps1 locus (middle panel), and the Slc5a8 locus (right panel). ATAC and M-ATAC in green, CTCF in purple and DNA methylation from merged M-ATAC, merged CTCF M-ChIP and WGBS (methylation from 0% in blue to 100% in red). A zoom-in of methylation at the highlighted region is shown at the bottom of each example. The Klf4 locus illustrates a region that has low methylation as detected by M-ATAC, CTCF M-ChIP, and WGBS. The Pisd-ps1 locus illustrates a region that has high methylation as detected by M-ATAC, CTCF M-ChIP, and WGBS. The Slc5a8 locus illustrates a region that has low methylation as detected by M-ATAC and high methylation as detected by WGBS. c Density plots of methylation from EpiMethyltag compared with WGBS. Only CpGs inside peaks and with at least five reads were considered. Top: average methylation of CpGs per M-ATAC peak in M-ATAC versus WGBS (Pearson correlation = 0.69, p value

    Techniques Used: Binding Assay, Genome Wide, Chromatin Immunoprecipitation, Generated, DNA Methylation Assay, Methylation

    M-ChIP enables analysis of DNA methylation binding by CTCF and KLF4. a Top: Schematic illustration representing an ATAC-seq peak with a CTCF motif and CTCF occupancy dependent on C2 and C12 methylation. Bottom: average profiles of M-ATAC (left) and CTCF M-ChIP (right) intensity at CpGs in a CTCF motif within M-ATAC peaks for the four groups of CpGs (group #1: 288 CpGs, group #2: 17133 CpGs, group #3 CpGs: 758, group #4: 25 CpGs). b top: CTCF motif from JASPAR database (MA0139.1). The 2 key CpG positions (C2 and C12) are indicated. Bottom: violin plots of methylation percentage from CTCF M-ChIP and WGBS, at C2 and C12 positions in the CTCF motif (MA0139.1). *** P = 1.02e−12 for C2 CTCF M-ChIP versus C12 CTCF M-ChIP (Wilcoxon test), ** P = 0.008 for C2 WGBS versus C12 WGBS (Wilcoxon test), *** P = 9e−12 for C2 CTCF M-ChIP versus C2 WGBS (Wilcoxon test, paired), *** P = 0.00075 for C12 CTCF M-ChIP versus C12 WGBS (Wilcoxon test, paired), * P = 0.023 for CTCF M-ChIP versus WGBS (logistic regression model). c Scatter plot showing the relationship between binding strength and CpG methylation within the KLF4 M-ChIP peaks (Pearson correlation = 0.25; bottom left corner: 5138 CpGs, top left corner: 578 CpGs, top right corner: 104 CpGs, bottom right corner: 60 CpGs). d Venn diagram showing the overlap between WT and mutant KLF4 M-ChIP peaks. e Top: Illustration of KLF4 motifs from the Jaspar database (MA0039.1 and MA0039.2). The black bar represents the potential CpGs present in the MA0039.2 motif. Bottom: histogram showing the relative distribution of KLF4 motifs in WT, mutant and common KLF4 M-ChIP peaks using FIMO from the MEME suite. Absolute numbers of each motif are indicated. f Heatmap showing M-ATAC signal intensity at KLF4 M-ChIP peaks that are specific to WT (1836 peaks), mutant (267 peaks), or common between both conditions (303 peaks). g Average cytosine methylation from M-ATAC in WT versus mutant KLF4 expressing cells in WT specific KLF4 M-ChIP peaks (Pearson correlation = 0.78, p value
    Figure Legend Snippet: M-ChIP enables analysis of DNA methylation binding by CTCF and KLF4. a Top: Schematic illustration representing an ATAC-seq peak with a CTCF motif and CTCF occupancy dependent on C2 and C12 methylation. Bottom: average profiles of M-ATAC (left) and CTCF M-ChIP (right) intensity at CpGs in a CTCF motif within M-ATAC peaks for the four groups of CpGs (group #1: 288 CpGs, group #2: 17133 CpGs, group #3 CpGs: 758, group #4: 25 CpGs). b top: CTCF motif from JASPAR database (MA0139.1). The 2 key CpG positions (C2 and C12) are indicated. Bottom: violin plots of methylation percentage from CTCF M-ChIP and WGBS, at C2 and C12 positions in the CTCF motif (MA0139.1). *** P = 1.02e−12 for C2 CTCF M-ChIP versus C12 CTCF M-ChIP (Wilcoxon test), ** P = 0.008 for C2 WGBS versus C12 WGBS (Wilcoxon test), *** P = 9e−12 for C2 CTCF M-ChIP versus C2 WGBS (Wilcoxon test, paired), *** P = 0.00075 for C12 CTCF M-ChIP versus C12 WGBS (Wilcoxon test, paired), * P = 0.023 for CTCF M-ChIP versus WGBS (logistic regression model). c Scatter plot showing the relationship between binding strength and CpG methylation within the KLF4 M-ChIP peaks (Pearson correlation = 0.25; bottom left corner: 5138 CpGs, top left corner: 578 CpGs, top right corner: 104 CpGs, bottom right corner: 60 CpGs). d Venn diagram showing the overlap between WT and mutant KLF4 M-ChIP peaks. e Top: Illustration of KLF4 motifs from the Jaspar database (MA0039.1 and MA0039.2). The black bar represents the potential CpGs present in the MA0039.2 motif. Bottom: histogram showing the relative distribution of KLF4 motifs in WT, mutant and common KLF4 M-ChIP peaks using FIMO from the MEME suite. Absolute numbers of each motif are indicated. f Heatmap showing M-ATAC signal intensity at KLF4 M-ChIP peaks that are specific to WT (1836 peaks), mutant (267 peaks), or common between both conditions (303 peaks). g Average cytosine methylation from M-ATAC in WT versus mutant KLF4 expressing cells in WT specific KLF4 M-ChIP peaks (Pearson correlation = 0.78, p value

    Techniques Used: Chromatin Immunoprecipitation, DNA Methylation Assay, Binding Assay, Methylation, CpG Methylation Assay, Mutagenesis, Expressing

    61) Product Images from "Expanding epigenomics to archived FFPE tissues: An evaluation of DNA repair methodologies"

    Article Title: Expanding epigenomics to archived FFPE tissues: An evaluation of DNA repair methodologies

    Journal: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology

    doi: 10.1158/1055-9965.EPI-14-0464

    Schematic of Experimental Parameters comparing FFPE DNA repair methods to FF DNA DNA (500ng) from FF tissue was processed according to Illumina Human Methylation instructions, including bisulfite modification followed by the standard Human Methylation processing protocol (Gold standard). Experimental conditions are separated for REPLI-g ligation (LIG) and Illumina Restore Kit (RES). LIG1 : the original Thirwell method using 500ng of genomic DNA processed by REPLI-g ligase and bisulfite modified (BS), and 4µl of bisulfite-modified DNA used for the starting material for the Illumina Human Methylation array kit. LIG2 : Thirwell method with output DNA increased to 8µl of bisulfite modified DNA, which is the same as used in the Restore Kit. LIG3 and LIG4 : 500ng and 250ng of genomic DNA, respectively, were bisulfite modified, processed by REPLI-g ligase and 8µl of material used for Illumina Methylation Array kit. RES1 : the Illumina Restore protocol using 500ng of genomic DNA, bisulfite modified and processed per Restore Kit protocol (including 8µl for Array steps). RES2 and RES3 : Technical replicates for Restore Kit using 250ng of genomic DNA.
    Figure Legend Snippet: Schematic of Experimental Parameters comparing FFPE DNA repair methods to FF DNA DNA (500ng) from FF tissue was processed according to Illumina Human Methylation instructions, including bisulfite modification followed by the standard Human Methylation processing protocol (Gold standard). Experimental conditions are separated for REPLI-g ligation (LIG) and Illumina Restore Kit (RES). LIG1 : the original Thirwell method using 500ng of genomic DNA processed by REPLI-g ligase and bisulfite modified (BS), and 4µl of bisulfite-modified DNA used for the starting material for the Illumina Human Methylation array kit. LIG2 : Thirwell method with output DNA increased to 8µl of bisulfite modified DNA, which is the same as used in the Restore Kit. LIG3 and LIG4 : 500ng and 250ng of genomic DNA, respectively, were bisulfite modified, processed by REPLI-g ligase and 8µl of material used for Illumina Methylation Array kit. RES1 : the Illumina Restore protocol using 500ng of genomic DNA, bisulfite modified and processed per Restore Kit protocol (including 8µl for Array steps). RES2 and RES3 : Technical replicates for Restore Kit using 250ng of genomic DNA.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Methylation, Modification, Ligation

    62) Product Images from "Suppression of indoleamine-2,3-dioxygenase 1 expression by promoter hypermethylation in ER-positive breast cancer"

    Article Title: Suppression of indoleamine-2,3-dioxygenase 1 expression by promoter hypermethylation in ER-positive breast cancer

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2016.1274477

    The IDO1 promoter is hypermethylated in ER-positive breast cancer. (A) Schematic representation of regulatory elements in the human IDO1 promoter. In an upstream region of active chromatin (ENCODE ChromHMM E027 and E028, enrichment of H3K27Ac, DNase hypersensitivity cluster) an interferon sensitive response element (ISRE) and a STAT1 binding site overlap with several CpG sites as can be seen in the WGBS data. One of these CpG sites— cg10262052—is covered by 450k arrays (GRCh 37/hg19 assembly). The localization of our IDO1 luciferase reporter gene construct is depicted in green. (B) The DNA methylation level of cg10262052 is significantly lower in ER-negative ( n = 99) compared with ER-positive ( n = 343) breast cancers (TCGA breast invasive carcinoma, Mann–Whitney U test, *** p
    Figure Legend Snippet: The IDO1 promoter is hypermethylated in ER-positive breast cancer. (A) Schematic representation of regulatory elements in the human IDO1 promoter. In an upstream region of active chromatin (ENCODE ChromHMM E027 and E028, enrichment of H3K27Ac, DNase hypersensitivity cluster) an interferon sensitive response element (ISRE) and a STAT1 binding site overlap with several CpG sites as can be seen in the WGBS data. One of these CpG sites— cg10262052—is covered by 450k arrays (GRCh 37/hg19 assembly). The localization of our IDO1 luciferase reporter gene construct is depicted in green. (B) The DNA methylation level of cg10262052 is significantly lower in ER-negative ( n = 99) compared with ER-positive ( n = 343) breast cancers (TCGA breast invasive carcinoma, Mann–Whitney U test, *** p

    Techniques Used: Binding Assay, Luciferase, Construct, DNA Methylation Assay, MANN-WHITNEY

    63) Product Images from "Epigenomic evolution in diffuse large B-cell lymphomas"

    Article Title: Epigenomic evolution in diffuse large B-cell lymphomas

    Journal: Nature Communications

    doi: 10.1038/ncomms7921

    Convergence of methylation patterns at relapse involves key genes and pathways. ( a ) A locus displayed decreased intra-tumour MH from diagnosis to relapse. T and C at CpG site indicate unmethylated and methylated CpG separately. The tumour cell population displayed diverse DNA methylation patterns at diagnosis and the diversity is complete loss at relapse. ( b ) Pathways over-represented with decreased intra-tumour MH genes. P values were 0.0005, 0.0005, 0.0015 and 0.0048 from top to bottom (hypergeometric tests). GO analyses were performed with iPAGE 20 . Known pathways in the GO 46 were used here. The background included around 24,000 genes from Refseq gene annotation. The red colour indicates (in log 10 ) the over-represented P values and the blue shows under-representation. ( c ) Correlations between the average intra-tumour MH derived from ERRBS or Bisulfite-PCR-MiSeq in ENGASE promoters. ( d ) Correlations between the average intra-tumour MH derived from ERRBS or Bisulfite-PCR-MiSeq in ECHDC3 promoters. In c , d , MiSeq based intra-tumour MH was calculated using the same analytical approach as the one used for ERRBS. Red and blue dots represent diagnosis and relapsed samples, respectively. P values were derived from correlation test (cor.test() function in R ).
    Figure Legend Snippet: Convergence of methylation patterns at relapse involves key genes and pathways. ( a ) A locus displayed decreased intra-tumour MH from diagnosis to relapse. T and C at CpG site indicate unmethylated and methylated CpG separately. The tumour cell population displayed diverse DNA methylation patterns at diagnosis and the diversity is complete loss at relapse. ( b ) Pathways over-represented with decreased intra-tumour MH genes. P values were 0.0005, 0.0005, 0.0015 and 0.0048 from top to bottom (hypergeometric tests). GO analyses were performed with iPAGE 20 . Known pathways in the GO 46 were used here. The background included around 24,000 genes from Refseq gene annotation. The red colour indicates (in log 10 ) the over-represented P values and the blue shows under-representation. ( c ) Correlations between the average intra-tumour MH derived from ERRBS or Bisulfite-PCR-MiSeq in ENGASE promoters. ( d ) Correlations between the average intra-tumour MH derived from ERRBS or Bisulfite-PCR-MiSeq in ECHDC3 promoters. In c , d , MiSeq based intra-tumour MH was calculated using the same analytical approach as the one used for ERRBS. Red and blue dots represent diagnosis and relapsed samples, respectively. P values were derived from correlation test (cor.test() function in R ).

    Techniques Used: Methylation, DNA Methylation Assay, Derivative Assay, Polymerase Chain Reaction

    64) Product Images from "Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype"

    Article Title: Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

    Journal: Respiratory Research

    doi: 10.1186/1465-9921-13-74

    The Thy-1 promoter becomes hemimethylated in hypoxic pulmonary fibroblasts. Human pulmonary fibroblasts were grown in hypoxia for 4 and 8 days. Genomic DNA was bisulfite treated and methylation specific PCR (MSPCR) using unmethylated (U) and methylated (M) specific primers were used to investigate the methylation status of the Thy-1 promoter. MSPCR revealed Thy-1 promoter hemimethylation in hypoxic cells (n = 3) that was not present in normoxic controls (n = 3).
    Figure Legend Snippet: The Thy-1 promoter becomes hemimethylated in hypoxic pulmonary fibroblasts. Human pulmonary fibroblasts were grown in hypoxia for 4 and 8 days. Genomic DNA was bisulfite treated and methylation specific PCR (MSPCR) using unmethylated (U) and methylated (M) specific primers were used to investigate the methylation status of the Thy-1 promoter. MSPCR revealed Thy-1 promoter hemimethylation in hypoxic cells (n = 3) that was not present in normoxic controls (n = 3).

    Techniques Used: Methylation, Polymerase Chain Reaction

    65) Product Images from "Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing"

    Article Title: Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr598

    Targeted bisulfite sequencing. ( a ) Illustrated are the steps involved in the preparation of biotinylated RNA capture probes (top left and right), whole-genome fragment input library (top middle) and hybrid selection-enriched output library (middle left and right). The captured DNA was treated with sodium bisulfite, amplified by PCR and sequenced using an Illumina GAIIx sequencer. ( b ) Examples of targeted bisulfite sequencing using solution hybrid selection. The tracks shown from the top to bottom are: sequences of the capture probes; the DNA methylation level at each CpG site derived from the bisulfite-sequencing reads; the sequencing depth at each CpG site; RefSeq genes; and annotated CGIs in the UCSC genome browser. Red and green colors indicate methylated and unmethylated-CpG sites, respectively. MCF7-1 and MCF7-2 are technical replicates. For each sample, two tracks (methylation level and read depth) are shown. ( c ). Distribution of normalized abundance for the captured targets shared among all samples. The x -axis is the normalized abundance of each captured target, which is calculated by dividing the counts of the target by the average counts of all targets. The y -axis is the fraction of probes with coverage equal to or greater than the normalized coverage.
    Figure Legend Snippet: Targeted bisulfite sequencing. ( a ) Illustrated are the steps involved in the preparation of biotinylated RNA capture probes (top left and right), whole-genome fragment input library (top middle) and hybrid selection-enriched output library (middle left and right). The captured DNA was treated with sodium bisulfite, amplified by PCR and sequenced using an Illumina GAIIx sequencer. ( b ) Examples of targeted bisulfite sequencing using solution hybrid selection. The tracks shown from the top to bottom are: sequences of the capture probes; the DNA methylation level at each CpG site derived from the bisulfite-sequencing reads; the sequencing depth at each CpG site; RefSeq genes; and annotated CGIs in the UCSC genome browser. Red and green colors indicate methylated and unmethylated-CpG sites, respectively. MCF7-1 and MCF7-2 are technical replicates. For each sample, two tracks (methylation level and read depth) are shown. ( c ). Distribution of normalized abundance for the captured targets shared among all samples. The x -axis is the normalized abundance of each captured target, which is calculated by dividing the counts of the target by the average counts of all targets. The y -axis is the fraction of probes with coverage equal to or greater than the normalized coverage.

    Techniques Used: Methylation Sequencing, Selection, Amplification, Polymerase Chain Reaction, DNA Methylation Assay, Derivative Assay, Sequencing, Methylation

    66) Product Images from "Genome-wide mapping of DNA methylation: a quantitative technology comparison"

    Article Title: Genome-wide mapping of DNA methylation: a quantitative technology comparison

    Journal: Nature biotechnology

    doi: 10.1038/nbt.1681

    Comparison of DNA methylation maps obtained with MeDIP, MethylCap, RRBS and Infinium DNA methylation maps were generated using MeDIP (first two tracks, in green), MethylCap (three tracks in blue, grey and red), RRBS (stacked blue tracks) and Infinium (single black track with percentage values) and converted into UCSC Genome Browser tracks. The screenshot shows the HOXA cluster in a human ES cell line (HUES6). Each track represents data from a single sequencing lane (MeDIP, MethylCap, RRBS) or microarray hybridization (Infinium). MeDIP and MethylCap data are visually similar to ChIP-seq data, with peaks in regions that exhibit high density of the target molecule (5-methyl-cytosine) and troughs in regions with low density of methylated cytosines. The height of the peaks represents the number of reads in each genomic interval, for each track normalized to the same genome-wide read count (note the twofold compressed scaling of the MethylCap tracks relative to the MeDIP tracks, which is indicative of higher dynamic range for MethylCap compared to MeDIP). RRBS gives rise to clusters of CpGs with absolute DNA methylation measurements, separated by regions that are not covered due to the reduced-representation property of the RRBS protocol. Each data point corresponds to the methylation level at a single CpG, and dark blue points indicate higher methylation levels than light blue points. Infinium data is represented in a similar way as the RRBS data, and the methylation levels at single CpGs are shown as percentage values. The three grey columns highlight regions that are illustrative of specific properties of the enrichment methods: (1) A promoter region that is CpG-poor and therefore not detectable by MeDIP or MethylCap – independent of its DNA methylation level; (2) a promoter region that contains many CpGs but low levels of DNA methylation, which also results in the absence of MeDIP and MethylCap peaks; and (3) a CpG island that exhibits a strong enrichment peak for both MeDIP and MethylCap although the RRBS data indicates that it is only partially methylated. For reference, the CpG density is indicated by stacked points (black) at the bottom of the diagram, and CpG islands (red) as well as known genes (blue) are listed as described previously 64 , 65 . All DNA methylation maps are available online as custom tracks for interactive visualization in the UCSC Genome Browser ( http://meth-benchmark.computational-epigenetics.org/ ).
    Figure Legend Snippet: Comparison of DNA methylation maps obtained with MeDIP, MethylCap, RRBS and Infinium DNA methylation maps were generated using MeDIP (first two tracks, in green), MethylCap (three tracks in blue, grey and red), RRBS (stacked blue tracks) and Infinium (single black track with percentage values) and converted into UCSC Genome Browser tracks. The screenshot shows the HOXA cluster in a human ES cell line (HUES6). Each track represents data from a single sequencing lane (MeDIP, MethylCap, RRBS) or microarray hybridization (Infinium). MeDIP and MethylCap data are visually similar to ChIP-seq data, with peaks in regions that exhibit high density of the target molecule (5-methyl-cytosine) and troughs in regions with low density of methylated cytosines. The height of the peaks represents the number of reads in each genomic interval, for each track normalized to the same genome-wide read count (note the twofold compressed scaling of the MethylCap tracks relative to the MeDIP tracks, which is indicative of higher dynamic range for MethylCap compared to MeDIP). RRBS gives rise to clusters of CpGs with absolute DNA methylation measurements, separated by regions that are not covered due to the reduced-representation property of the RRBS protocol. Each data point corresponds to the methylation level at a single CpG, and dark blue points indicate higher methylation levels than light blue points. Infinium data is represented in a similar way as the RRBS data, and the methylation levels at single CpGs are shown as percentage values. The three grey columns highlight regions that are illustrative of specific properties of the enrichment methods: (1) A promoter region that is CpG-poor and therefore not detectable by MeDIP or MethylCap – independent of its DNA methylation level; (2) a promoter region that contains many CpGs but low levels of DNA methylation, which also results in the absence of MeDIP and MethylCap peaks; and (3) a CpG island that exhibits a strong enrichment peak for both MeDIP and MethylCap although the RRBS data indicates that it is only partially methylated. For reference, the CpG density is indicated by stacked points (black) at the bottom of the diagram, and CpG islands (red) as well as known genes (blue) are listed as described previously 64 , 65 . All DNA methylation maps are available online as custom tracks for interactive visualization in the UCSC Genome Browser ( http://meth-benchmark.computational-epigenetics.org/ ).

    Techniques Used: DNA Methylation Assay, Methylated DNA Immunoprecipitation, Generated, Sequencing, Microarray, Hybridization, Chromatin Immunoprecipitation, Methylation, Genome Wide

    Detection of differentially methylated regions with MeDIP, MethylCap and RRBS Average DNA methylation measurements were calculated for each CpG island and compared between two human ES cell lines (HUES6 and HUES8). Total read frequencies are shown for MeDIP (panel A) and MethylCap (panel B), and mean DNA methylation levels are shown for RRBS (panel C). Regions with insufficient sequencing coverage were excluded. The Venn diagram (panel D) displays the total number and mutual overlap of differentially methylated CpG islands that could be identified by each method. CpG islands were classified as hypermethylated or hypomethylated (depending on the directionality of the difference) if the absolute DNA methylation difference exceeded 20% (for RRBS) or if there was at least a twofold difference in read number between the two samples (for MeDIP and MethylCap) – but only if Fisher’s exact test with multiple-testing correction gave rise to an estimated false-discovery rate of differential DNA methylation that was less than 0.1.
    Figure Legend Snippet: Detection of differentially methylated regions with MeDIP, MethylCap and RRBS Average DNA methylation measurements were calculated for each CpG island and compared between two human ES cell lines (HUES6 and HUES8). Total read frequencies are shown for MeDIP (panel A) and MethylCap (panel B), and mean DNA methylation levels are shown for RRBS (panel C). Regions with insufficient sequencing coverage were excluded. The Venn diagram (panel D) displays the total number and mutual overlap of differentially methylated CpG islands that could be identified by each method. CpG islands were classified as hypermethylated or hypomethylated (depending on the directionality of the difference) if the absolute DNA methylation difference exceeded 20% (for RRBS) or if there was at least a twofold difference in read number between the two samples (for MeDIP and MethylCap) – but only if Fisher’s exact test with multiple-testing correction gave rise to an estimated false-discovery rate of differential DNA methylation that was less than 0.1.

    Techniques Used: Methylation, Methylated DNA Immunoprecipitation, DNA Methylation Assay, Sequencing

    Quantification of DNA methylation with MeDIP, MethylCap and RRBS Absolute DNA methylation levels were calculated from the data obtained by MeDIP (panel A), MethylCap (panel B) and RRBS (panel C), respectively, and compared to DNA methylation levels determined by the Infinium assay. For MeDIP and MethylCap, sequencing reads were counted in 1-kilobase regions surrounding each CpG that is interrogated by the Infinium assay, and a regression model was used to infer absolute DNA methylation levels. Scatterplots and correlation coefficients were calculated on a test set that was not used for model fitting or feature selection. For RRBS, the DNA methylation level was determined as the percentage of methylated CpGs within 200 basepairs surrounding each CpG that is interrogated by the Infinium assay. Data shown are for the HUES6 human ES cell line, and regions that did not have sufficient sequencing coverage were excluded.
    Figure Legend Snippet: Quantification of DNA methylation with MeDIP, MethylCap and RRBS Absolute DNA methylation levels were calculated from the data obtained by MeDIP (panel A), MethylCap (panel B) and RRBS (panel C), respectively, and compared to DNA methylation levels determined by the Infinium assay. For MeDIP and MethylCap, sequencing reads were counted in 1-kilobase regions surrounding each CpG that is interrogated by the Infinium assay, and a regression model was used to infer absolute DNA methylation levels. Scatterplots and correlation coefficients were calculated on a test set that was not used for model fitting or feature selection. For RRBS, the DNA methylation level was determined as the percentage of methylated CpGs within 200 basepairs surrounding each CpG that is interrogated by the Infinium assay. Data shown are for the HUES6 human ES cell line, and regions that did not have sufficient sequencing coverage were excluded.

    Techniques Used: DNA Methylation Assay, Methylated DNA Immunoprecipitation, Sequencing, Selection, Methylation

    Genomic coverage of MeDIP, MethylCap, RRBS and Infinium Genomic coverage was quantified by the number of DNA methylation measurements that overlap with CpG islands (top row), gene promoters (center row) and a 1-kilobase tiling of the genome (bottom row). For MeDIP and MethylCap, the number of measurements is equal to the number of unique sequencing reads that fall inside each region. For RRBS, it refers to the number of valid DNA methylation measurements at CpGs within each region (one RRBS sequencing read typically yields one measurement, but can also give rise to more than one measurement if it contains several CpGs). For Infinium, the number of measurements is equal to the number of CpGs within each region that are present on the HumanMethylation27 microarray. CpG islands were calculated using CgiHunter ( http://cgihunter.bioinf.mpi-inf.mpg.de/ ), requiring a minimum CpG observed vs. expected ratio of 0.6, a minimum GC content of 0.5 and a minimum length of 700 basepairs 64 . Promoter regions were calculated based on Ensembl gene annotations, such that the region starts one kilo-base upstream of the annotated transcription start site (TSS) and extends to one kilobase downstream of the TSS. The genomic tiling was obtained by sliding a 1-kilobase window through the genome such that each tile starts at the position where the previous tile ends. No repeat-masking was performed for any of the three types of genomic regions. Data are shown for the HUES6 human ES cell line.
    Figure Legend Snippet: Genomic coverage of MeDIP, MethylCap, RRBS and Infinium Genomic coverage was quantified by the number of DNA methylation measurements that overlap with CpG islands (top row), gene promoters (center row) and a 1-kilobase tiling of the genome (bottom row). For MeDIP and MethylCap, the number of measurements is equal to the number of unique sequencing reads that fall inside each region. For RRBS, it refers to the number of valid DNA methylation measurements at CpGs within each region (one RRBS sequencing read typically yields one measurement, but can also give rise to more than one measurement if it contains several CpGs). For Infinium, the number of measurements is equal to the number of CpGs within each region that are present on the HumanMethylation27 microarray. CpG islands were calculated using CgiHunter ( http://cgihunter.bioinf.mpi-inf.mpg.de/ ), requiring a minimum CpG observed vs. expected ratio of 0.6, a minimum GC content of 0.5 and a minimum length of 700 basepairs 64 . Promoter regions were calculated based on Ensembl gene annotations, such that the region starts one kilo-base upstream of the annotated transcription start site (TSS) and extends to one kilobase downstream of the TSS. The genomic tiling was obtained by sliding a 1-kilobase window through the genome such that each tile starts at the position where the previous tile ends. No repeat-masking was performed for any of the three types of genomic regions. Data are shown for the HUES6 human ES cell line.

    Techniques Used: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Sequencing, Microarray

    67) Product Images from "Semiconductor-based sequencing of genome-wide DNA methylation states"

    Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

    Journal: Epigenetics

    doi: 10.1080/15592294.2014.1003747

    Scalability of Ion Torrent-compatible MeDIP-Seq protocol. (a) Global 5-methylcytosine (5mC) content measured as a percentage of the genome in DNA from WT and DKO cells determined by an ELISA assay. (b) Number of peaks identified by MACS v2.1.0 software 64 from MeDIP-Seq data in WT and DKO cells. (c) Sequence coverage of genome-wide CpGs for WT and DKO cells on the Ion Torrent Proton sequencer calculated using MEDIPS v1.14.0 software. 39 Pie charts illustrate the fraction of CpGs covered by the indicated reads according to their fold-coverage in relation to the total genomic CpG content from MeDIP-Seq libraries of the indicated sample sequenced on the Proton with the total number of non-redundant mapped reads in parentheses. (d) Sequence coverage of genome-wide CpGs for WT and DKO cells in MeDIP-Seq on the Ion Torrent PGM sequencer. Data displayed as in c . (e) Scaled chromosomal view of the 5mC MeDIP-Seq enrichment of methylation over the MX1 gene in WT and DKO cells sequenced on the 2 different Ion Torrent sequencers as indicated. MeDIP-Seq data are displayed as RPM (Reads Per Million mapped reads) below the RefSeq annotation of the gene (blue lines). Methylation enrichment is displayed in gray, with red vertical lines corresponding to areas containing CpGs. Chromosome position and gene orientation are displayed.
    Figure Legend Snippet: Scalability of Ion Torrent-compatible MeDIP-Seq protocol. (a) Global 5-methylcytosine (5mC) content measured as a percentage of the genome in DNA from WT and DKO cells determined by an ELISA assay. (b) Number of peaks identified by MACS v2.1.0 software 64 from MeDIP-Seq data in WT and DKO cells. (c) Sequence coverage of genome-wide CpGs for WT and DKO cells on the Ion Torrent Proton sequencer calculated using MEDIPS v1.14.0 software. 39 Pie charts illustrate the fraction of CpGs covered by the indicated reads according to their fold-coverage in relation to the total genomic CpG content from MeDIP-Seq libraries of the indicated sample sequenced on the Proton with the total number of non-redundant mapped reads in parentheses. (d) Sequence coverage of genome-wide CpGs for WT and DKO cells in MeDIP-Seq on the Ion Torrent PGM sequencer. Data displayed as in c . (e) Scaled chromosomal view of the 5mC MeDIP-Seq enrichment of methylation over the MX1 gene in WT and DKO cells sequenced on the 2 different Ion Torrent sequencers as indicated. MeDIP-Seq data are displayed as RPM (Reads Per Million mapped reads) below the RefSeq annotation of the gene (blue lines). Methylation enrichment is displayed in gray, with red vertical lines corresponding to areas containing CpGs. Chromosome position and gene orientation are displayed.

    Techniques Used: Methylated DNA Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Magnetic Cell Separation, Software, Sequencing, Genome Wide, Methylation

    Validation of Ion Torrent-compatible MeDIP-Seq data by integration with the single-nucleotide resolution 450K array. ( a ) Histogram of the frequency and distribution of CpG cytosine methylation levels of probes on the 450K array in HCT116-WT (green) and -DKO (orange) cells stratified into categories based on their percentage of methylation. ( b ) Percentage of CpGs on the 450K array (black) overlapping with those underlying MeDIP-Seq peaks (white) of WT cells. ( c ) Distribution of methylation levels measured in WT cells of all probes on the 450K array that integrate with MeDIP-Seq peaks. ( d ) Distribution of methylation levels measured in DKO cells of all probes on the 450K array that integrate with MeDIP-Seq peaks in WT cells. ( e ) Scaled chromosomal view of the MeDIP-Seq enrichment of methylation over the ICR of the SNRPN gene. MeDIP-Seq from WT and DKO cells are displayed as indicated below the RefSeq annotation of SNRPN/SNURF (blue lines) and a CpG island (green). CpG methylation data of each 450K array probe over this region from WT and DKO cells are shown to scale as colored bars representing the degree of methylation (blue, low-; violet, intermediate-; and orange, high-levels of methylation). Percent methylation (calculated from β-values) of these CpGs is shown under each bar. The expanded region below these values shows a scaled depiction of CpG methylation of alleles sequenced after bisulfite-PCR in WT and DKO cells as indicated. Each circle represents a CpG cytosine in the amplicon sequenced, the color of which depicts its methylation state (white, unmethylated; black, methylated). *, the CpG on the 450K array also covered by the bisulfite-PCR amplicon. ( f ) The average expression of SNURF is significantly elevated in DKO compared to WT cells. Average expression values and s.e.m. from triplicate qPCR are shown. ( g ) DNA methylation over the SNURF promoter region is significantly higher in WT compared to DKO cells. Using bisulfite-PCR sequencing data described in e , percent methylation was calculated from the sum of methylated CpGs over the total CpGs evaluated. Average methylation values and s.e.m from individual clones are shown. P -values shown in f and g were calculated using 2-sided Student's t -test.
    Figure Legend Snippet: Validation of Ion Torrent-compatible MeDIP-Seq data by integration with the single-nucleotide resolution 450K array. ( a ) Histogram of the frequency and distribution of CpG cytosine methylation levels of probes on the 450K array in HCT116-WT (green) and -DKO (orange) cells stratified into categories based on their percentage of methylation. ( b ) Percentage of CpGs on the 450K array (black) overlapping with those underlying MeDIP-Seq peaks (white) of WT cells. ( c ) Distribution of methylation levels measured in WT cells of all probes on the 450K array that integrate with MeDIP-Seq peaks. ( d ) Distribution of methylation levels measured in DKO cells of all probes on the 450K array that integrate with MeDIP-Seq peaks in WT cells. ( e ) Scaled chromosomal view of the MeDIP-Seq enrichment of methylation over the ICR of the SNRPN gene. MeDIP-Seq from WT and DKO cells are displayed as indicated below the RefSeq annotation of SNRPN/SNURF (blue lines) and a CpG island (green). CpG methylation data of each 450K array probe over this region from WT and DKO cells are shown to scale as colored bars representing the degree of methylation (blue, low-; violet, intermediate-; and orange, high-levels of methylation). Percent methylation (calculated from β-values) of these CpGs is shown under each bar. The expanded region below these values shows a scaled depiction of CpG methylation of alleles sequenced after bisulfite-PCR in WT and DKO cells as indicated. Each circle represents a CpG cytosine in the amplicon sequenced, the color of which depicts its methylation state (white, unmethylated; black, methylated). *, the CpG on the 450K array also covered by the bisulfite-PCR amplicon. ( f ) The average expression of SNURF is significantly elevated in DKO compared to WT cells. Average expression values and s.e.m. from triplicate qPCR are shown. ( g ) DNA methylation over the SNURF promoter region is significantly higher in WT compared to DKO cells. Using bisulfite-PCR sequencing data described in e , percent methylation was calculated from the sum of methylated CpGs over the total CpGs evaluated. Average methylation values and s.e.m from individual clones are shown. P -values shown in f and g were calculated using 2-sided Student's t -test.

    Techniques Used: Methylated DNA Immunoprecipitation, Methylation, CpG Methylation Assay, Polymerase Chain Reaction, Amplification, Expressing, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Sequencing, Clone Assay

    Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.
    Figure Legend Snippet: Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.

    Techniques Used: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Genome Wide, Isolation, Sonication, Sequencing, Methylation, Immunoprecipitation, Agarose Gel Electrophoresis, Amplification, Concentration Assay, Chromatin Immunoprecipitation, Electron Microscopy, Software

    Ion Torrent-compatible MeDIP-Seq detects expected differences in DNA methylation between DNMT -proficient and - deficient cells. Distribution of 5mC methylation over the indicated bp window of all RefSeq annotated TSS ( a ), gene bodies ( b ), CpG islands ( c ), and exons ( d ). Methylation of WT (green line) and DKO (orange line) HCT116 cells are displayed as mean RPM values with s.e.m. indicated as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between WT and DKO methylation calculated using a one-sided KS test ( P -values shown). In c and d , a KS test was performed using data over the non-shaded areas as well ( P -values shown).
    Figure Legend Snippet: Ion Torrent-compatible MeDIP-Seq detects expected differences in DNA methylation between DNMT -proficient and - deficient cells. Distribution of 5mC methylation over the indicated bp window of all RefSeq annotated TSS ( a ), gene bodies ( b ), CpG islands ( c ), and exons ( d ). Methylation of WT (green line) and DKO (orange line) HCT116 cells are displayed as mean RPM values with s.e.m. indicated as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between WT and DKO methylation calculated using a one-sided KS test ( P -values shown). In c and d , a KS test was performed using data over the non-shaded areas as well ( P -values shown).

    Techniques Used: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation

    Ion Torrent-compatible MeDIP-Seq confirms a role for DNA methylation in alternative splicing. (a) Distribution of 5mC methylation over alternatively spliced exons (ASE) flanked by constitutively spliced exons (5’ or 3’ exons) in WT and DKO cells. Mean RPM values are displayed over ± 500 bp windows relative to the splice acceptor and donor sites at each of the exons as indicated, with s.e.m. depicted by shaded areas for WT (green) and DKO (orange) profiles. A schematic representation of cassette exons is shown, with the number of exons aberrantly included (blue) and excluded (gray) in WT vs. DKO cells indicated. P- values shown were calculated using one-sided KS test. (b) The ASE of the FAM204A gene (exon 2) is aberrantly excluded in DKO compared to WT cells. Schematic shows the 2 known isoforms for the FAM204A gene (blue bars depict the first 3 exons of the gene in the orientation indicated), under which a Sashimi plot generated by MISO analysis of RNA-Seq data measured as RPKM in WT and DKO cells illustrates the number of exon-exon junction reads as indicated to infer isoform expression. The left graphs show the MISO calculated distribution of a percent exon inclusion score (Psi-value; 95% confidence intervals in brackets) for the FAM204A ASE from WT (top) and DKO (bottom) RNA-Seq data. (c) Demethylation of the FAM204A ASE correlates with its aberrant exclusion. Scaled chromosomal view at the FAM204A gene region (ASE highlighted in yellow) of the indicated WT and DKO distribution of RNA-Seq (top pair) and DNA methylation (bottom pair) data displayed as RPKM. The first 3 exons of the gene are represented (blue bars) with a CpG island in the promoter region indicated (green bar). In the MeDIP-Seq data, red vertical lines correspond to areas containing CpGs.
    Figure Legend Snippet: Ion Torrent-compatible MeDIP-Seq confirms a role for DNA methylation in alternative splicing. (a) Distribution of 5mC methylation over alternatively spliced exons (ASE) flanked by constitutively spliced exons (5’ or 3’ exons) in WT and DKO cells. Mean RPM values are displayed over ± 500 bp windows relative to the splice acceptor and donor sites at each of the exons as indicated, with s.e.m. depicted by shaded areas for WT (green) and DKO (orange) profiles. A schematic representation of cassette exons is shown, with the number of exons aberrantly included (blue) and excluded (gray) in WT vs. DKO cells indicated. P- values shown were calculated using one-sided KS test. (b) The ASE of the FAM204A gene (exon 2) is aberrantly excluded in DKO compared to WT cells. Schematic shows the 2 known isoforms for the FAM204A gene (blue bars depict the first 3 exons of the gene in the orientation indicated), under which a Sashimi plot generated by MISO analysis of RNA-Seq data measured as RPKM in WT and DKO cells illustrates the number of exon-exon junction reads as indicated to infer isoform expression. The left graphs show the MISO calculated distribution of a percent exon inclusion score (Psi-value; 95% confidence intervals in brackets) for the FAM204A ASE from WT (top) and DKO (bottom) RNA-Seq data. (c) Demethylation of the FAM204A ASE correlates with its aberrant exclusion. Scaled chromosomal view at the FAM204A gene region (ASE highlighted in yellow) of the indicated WT and DKO distribution of RNA-Seq (top pair) and DNA methylation (bottom pair) data displayed as RPKM. The first 3 exons of the gene are represented (blue bars) with a CpG island in the promoter region indicated (green bar). In the MeDIP-Seq data, red vertical lines correspond to areas containing CpGs.

    Techniques Used: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation, Generated, RNA Sequencing Assay, Expressing

    68) Product Images from "ATXR5 and ATXR6 are novel H3K27 monomethyltransferases required for chromatin structure and gene silencing"

    Article Title: ATXR5 and ATXR6 are novel H3K27 monomethyltransferases required for chromatin structure and gene silencing

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1611

    Mutations in atxr5 and atxr6 do not affect H3K9 dimethylation or DNA methylation. ( a ) Leaf interphase nuclei were stained with DAPI and analyzed for immunoflorescence with anti-H3K9me2 antibodies. Scale bar = 5 μm. (b, d ) DNA methylation analysis by (b) locus-specific ( Ta3 and CACTA ) and (d) genome-wide BS-Seq. Black and white bars represent wild type and atxr5 atxr6 , respectively. ( c ) ChIP analysis of repetitive elements using H3K9me2 antibodies. Black and white bars indicate relative levels of immunoprecipitated DNA normalized to ACTIN , as determined by real-time PCR, from wild type and atxr5 atxr6 , respectively. Grey bars represent no antibody controls. The data are presented as mean ± SEM for three individual experiments. ( e ) Distribution of methylation along the five Arabidopsis chromosomes. ( f ) Average methylation levels within protein coding genes. ( g ) Average methylation levels within pseudogenes and transposons. (e–g) A horizontal blue line indicates zero percent methylation. Panels on the left correspond to wild type and panels on the right correspond to atxr5 atxr6 double mutant. CG methylation is indicated by green lines, CHG methylation is indicated by yellow lines, and CHH methylation is indicated by red lines. (e) Vertical blue lines are used to separate different chromosomes. (f, g) Vertical blue lines mark the boundaries between upstream regions and gene bodies and between gene bodies and downstream regions. ( h) Leaf interphase nuclei of wild-type plants and kyp suvh5 suvh6 triple mutants were stained with DAPI and analyzed for immunofluorescence with anti-H3K27me1. Scale bar = 5 μm.
    Figure Legend Snippet: Mutations in atxr5 and atxr6 do not affect H3K9 dimethylation or DNA methylation. ( a ) Leaf interphase nuclei were stained with DAPI and analyzed for immunoflorescence with anti-H3K9me2 antibodies. Scale bar = 5 μm. (b, d ) DNA methylation analysis by (b) locus-specific ( Ta3 and CACTA ) and (d) genome-wide BS-Seq. Black and white bars represent wild type and atxr5 atxr6 , respectively. ( c ) ChIP analysis of repetitive elements using H3K9me2 antibodies. Black and white bars indicate relative levels of immunoprecipitated DNA normalized to ACTIN , as determined by real-time PCR, from wild type and atxr5 atxr6 , respectively. Grey bars represent no antibody controls. The data are presented as mean ± SEM for three individual experiments. ( e ) Distribution of methylation along the five Arabidopsis chromosomes. ( f ) Average methylation levels within protein coding genes. ( g ) Average methylation levels within pseudogenes and transposons. (e–g) A horizontal blue line indicates zero percent methylation. Panels on the left correspond to wild type and panels on the right correspond to atxr5 atxr6 double mutant. CG methylation is indicated by green lines, CHG methylation is indicated by yellow lines, and CHH methylation is indicated by red lines. (e) Vertical blue lines are used to separate different chromosomes. (f, g) Vertical blue lines mark the boundaries between upstream regions and gene bodies and between gene bodies and downstream regions. ( h) Leaf interphase nuclei of wild-type plants and kyp suvh5 suvh6 triple mutants were stained with DAPI and analyzed for immunofluorescence with anti-H3K27me1. Scale bar = 5 μm.

    Techniques Used: DNA Methylation Assay, Staining, Genome Wide, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Methylation, Mutagenesis, Immunofluorescence

    atxr5 atxr6 mutations lead to disruption of constitutive heterochromatin, reduced H3K27 monomethylation, and reactivation of silenced elements. (a) Leaf interphase nuclei were stained with DAPI and analyzed for immunofluorescence with an anti-H3K27me1 antibody. Approximately 65% of atxr5 atxr6 nuclei show severe (a, upper panel) or moderate (a, lower panel) chromocenter decondensation and reduced H3K27me1 staining. Scale bar = 5 μm. ( b) FISH analysis of leaf interphase nuclei using a 180-bp centromeric repeat probe. The DNA was counterstained with DAPI. Scale bar = 5 μm. ( c ) Semi-quantitative RT-PCR analysis of heterochromatic elements in Col, atxr5 , atxr6, and atxr5 atxr6. UBQ was used as a constitutively expressed control. ( d ) ChIP analysis of repetitive elements using H3K27me1 antibodies. Black and white bars indicate relative levels of immunoprecipitated DNA normalized to ACTIN , as determined by real-time PCR, from wild type and atxr5 atxr6 leaves, respectively. Grey bars represent no antibody controls. The data are presented as mean ± SEM for three individual experiments.
    Figure Legend Snippet: atxr5 atxr6 mutations lead to disruption of constitutive heterochromatin, reduced H3K27 monomethylation, and reactivation of silenced elements. (a) Leaf interphase nuclei were stained with DAPI and analyzed for immunofluorescence with an anti-H3K27me1 antibody. Approximately 65% of atxr5 atxr6 nuclei show severe (a, upper panel) or moderate (a, lower panel) chromocenter decondensation and reduced H3K27me1 staining. Scale bar = 5 μm. ( b) FISH analysis of leaf interphase nuclei using a 180-bp centromeric repeat probe. The DNA was counterstained with DAPI. Scale bar = 5 μm. ( c ) Semi-quantitative RT-PCR analysis of heterochromatic elements in Col, atxr5 , atxr6, and atxr5 atxr6. UBQ was used as a constitutively expressed control. ( d ) ChIP analysis of repetitive elements using H3K27me1 antibodies. Black and white bars indicate relative levels of immunoprecipitated DNA normalized to ACTIN , as determined by real-time PCR, from wild type and atxr5 atxr6 leaves, respectively. Grey bars represent no antibody controls. The data are presented as mean ± SEM for three individual experiments.

    Techniques Used: Staining, Immunofluorescence, Fluorescence In Situ Hybridization, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    69) Product Images from "Oxidized low-density lipoprotein is a common risk factor for cardiovascular diseases and gastroenterological cancers via epigenomical regulation of microRNA-210"

    Article Title: Oxidized low-density lipoprotein is a common risk factor for cardiovascular diseases and gastroenterological cancers via epigenomical regulation of microRNA-210

    Journal: Oncotarget

    doi:

    oxLDL effects on DNA demethylation in the miR-210 gene promoter of HASMCs A. CpG sites and the HIF-1α-binding site predictions in the miR-210 gene promoter. R1 to R3 means the regions used to detect methylation status by the BSP assay. B. DNA methylation changes in the miR-210 gene promoter according to the BSP assay. The HIF-1α-binding site is indicated by an arrow. Each row and circle respectively means one single sequencing reaction and one CpG site. The empty and solid circles respectively mean the un-methyl and methyl CpG site. C. Quantitative results for figure (B). D. DNA methylation changes in the miR-210 gene promoter by the MSP assay. After HASMCs were treated with 40 μg/ml oxLDL for 48 h, genomic DNA was extracted. The methylation status was determined by both the BSP and MSP assays. E. DNA methylation effects on oxLDL-mediated miR-210 gene promoter activity. The vector containing the miR-210 promoter was methylated by methylase in vitro and transfected into cells. After treatment with 40 μg/ml oxLDL for 48 h, luciferase activity was measured in triplicate experiments. Data are means ± SD of three experiments. * P
    Figure Legend Snippet: oxLDL effects on DNA demethylation in the miR-210 gene promoter of HASMCs A. CpG sites and the HIF-1α-binding site predictions in the miR-210 gene promoter. R1 to R3 means the regions used to detect methylation status by the BSP assay. B. DNA methylation changes in the miR-210 gene promoter according to the BSP assay. The HIF-1α-binding site is indicated by an arrow. Each row and circle respectively means one single sequencing reaction and one CpG site. The empty and solid circles respectively mean the un-methyl and methyl CpG site. C. Quantitative results for figure (B). D. DNA methylation changes in the miR-210 gene promoter by the MSP assay. After HASMCs were treated with 40 μg/ml oxLDL for 48 h, genomic DNA was extracted. The methylation status was determined by both the BSP and MSP assays. E. DNA methylation effects on oxLDL-mediated miR-210 gene promoter activity. The vector containing the miR-210 promoter was methylated by methylase in vitro and transfected into cells. After treatment with 40 μg/ml oxLDL for 48 h, luciferase activity was measured in triplicate experiments. Data are means ± SD of three experiments. * P

    Techniques Used: Binding Assay, Methylation, PCR-BSP Assay, DNA Methylation Assay, Sequencing, MSP Assay, Activity Assay, Plasmid Preparation, In Vitro, Transfection, Luciferase

    A high-fat diet reduces DNA methylation levels in the miR-210 promoter in vivo A. CpG island (gray color) and HIF-1α-binding site predictions in the mouse miR-210 gene promoter. Suitable primers were designed to amplify the CpG region containing the HIF-1α-binding site. B. The high-fat diet effects on DNA methylation in the mouse miR-210 gene promoter. The arrow indicates the HIF-1α-binding site. The N1 ∼ 3 and N4 ∼ 6 respectively means the serial number of control and high-fat diet-fed mouse. Each row and circle respectively means one single sequencing reaction and one CpG site. The empty and solid circles respectively mean the un-methyl and methyl CpG site. C. Quantitative results for. After being fed a high-fat diet, genomic DNA was extracted from the aorta of an APOE−/− mouse. The methylation status was measured by the BSP assay. D. The high-fat diet effects on miR-210 levels. Total RNA was extracted from aortas of mice fed a chow or high-fat diet, and miR-210 levels were measured by a qPCR. Data are means ± SD of three experiments. * P
    Figure Legend Snippet: A high-fat diet reduces DNA methylation levels in the miR-210 promoter in vivo A. CpG island (gray color) and HIF-1α-binding site predictions in the mouse miR-210 gene promoter. Suitable primers were designed to amplify the CpG region containing the HIF-1α-binding site. B. The high-fat diet effects on DNA methylation in the mouse miR-210 gene promoter. The arrow indicates the HIF-1α-binding site. The N1 ∼ 3 and N4 ∼ 6 respectively means the serial number of control and high-fat diet-fed mouse. Each row and circle respectively means one single sequencing reaction and one CpG site. The empty and solid circles respectively mean the un-methyl and methyl CpG site. C. Quantitative results for. After being fed a high-fat diet, genomic DNA was extracted from the aorta of an APOE−/− mouse. The methylation status was measured by the BSP assay. D. The high-fat diet effects on miR-210 levels. Total RNA was extracted from aortas of mice fed a chow or high-fat diet, and miR-210 levels were measured by a qPCR. Data are means ± SD of three experiments. * P

    Techniques Used: DNA Methylation Assay, In Vivo, Binding Assay, Sequencing, Methylation, PCR-BSP Assay, Mouse Assay, Real-time Polymerase Chain Reaction

    70) Product Images from "MTHFD1 controls DNA methylation in Arabidopsis"

    Article Title: MTHFD1 controls DNA methylation in Arabidopsis

    Journal: Nature Communications

    doi: 10.1038/ncomms11640

    SDCpro-GFP expression and DNA demethylation caused by R175Q mutation in MTHFD1 . ( a ) GFP fluorescence micrographs of WT, #162 M2, MTHFD1/ mthfd1-2 F1 and #162/ mthfd1-2 F1 seedlings. F1 are progeny of #162 M2 x MTHFD1/mthfd1-2 . Dashed boxes indicate magnified areas shown in lower panels. Inlets show bright-field images. ( b ) Gene structure, positions of mutations and conserved domains of MTHFD1. The EMS mutation in #162 lead to a R175Q substitution of a conserved residue required for NADP binding 28 . ( c ) PCR-based genotype analysis of 13 F1 seedlings and two control samples. Arrowheads mark bands corresponding to WT/ mthfd1-1 (upper) and mthfd1-2 (lower). The mthfd1-2 allele co-segregates with GFP fluorescence in F1 (+: present, −: absent). L, ladder. ( d ) Habit of different genotype plants 20 days after germination. Scale bar, 10 mm. ( e ) DNA blot analysis of non-CG methylation at the MEA-ISR locus. Genomic DNA was digested with methylation-sensitive MspI; upper and lower bands correspond to methylated (m) and unmethylated (u) fragments, respectively. Ratios of band intensities for each lane are shown under the gel image. ( f ) Levels of non-CG methylation at the AtSN1 locus by quantitative chop PCR analysis of genomic DNA after digestion with methylation-sensitive HaeIII relative to undigested DNA. Mean values±s.d. ( n =3). Different letters above bars indicate significant differences between pairwise comparisons by Student's t -test ( P
    Figure Legend Snippet: SDCpro-GFP expression and DNA demethylation caused by R175Q mutation in MTHFD1 . ( a ) GFP fluorescence micrographs of WT, #162 M2, MTHFD1/ mthfd1-2 F1 and #162/ mthfd1-2 F1 seedlings. F1 are progeny of #162 M2 x MTHFD1/mthfd1-2 . Dashed boxes indicate magnified areas shown in lower panels. Inlets show bright-field images. ( b ) Gene structure, positions of mutations and conserved domains of MTHFD1. The EMS mutation in #162 lead to a R175Q substitution of a conserved residue required for NADP binding 28 . ( c ) PCR-based genotype analysis of 13 F1 seedlings and two control samples. Arrowheads mark bands corresponding to WT/ mthfd1-1 (upper) and mthfd1-2 (lower). The mthfd1-2 allele co-segregates with GFP fluorescence in F1 (+: present, −: absent). L, ladder. ( d ) Habit of different genotype plants 20 days after germination. Scale bar, 10 mm. ( e ) DNA blot analysis of non-CG methylation at the MEA-ISR locus. Genomic DNA was digested with methylation-sensitive MspI; upper and lower bands correspond to methylated (m) and unmethylated (u) fragments, respectively. Ratios of band intensities for each lane are shown under the gel image. ( f ) Levels of non-CG methylation at the AtSN1 locus by quantitative chop PCR analysis of genomic DNA after digestion with methylation-sensitive HaeIII relative to undigested DNA. Mean values±s.d. ( n =3). Different letters above bars indicate significant differences between pairwise comparisons by Student's t -test ( P

    Techniques Used: Expressing, Mutagenesis, Fluorescence, Binding Assay, Polymerase Chain Reaction, Methylation, Microelectrode Array

    71) Product Images from "IQGAP1 and IQGAP2 are Reciprocally Altered in Hepatocellular Carcinoma"

    Article Title: IQGAP1 and IQGAP2 are Reciprocally Altered in Hepatocellular Carcinoma

    Journal: BMC Gastroenterology

    doi: 10.1186/1471-230X-10-125

    The Iqgap2 promoter is not hypermethylated in hepatocellular carcinoma . The degree of methylation for each CpG site was expressed as a percentage of methylated cytosines over the sum of total cytosines. The data are shown for 25 CpG sites and grouped by tissue type. Genomic DNA from the Kato III human gastric cancer cell line, which is known to have high methylation levels of the Iqgap2 gene promoter [ 23 ], was used as a positive control. The boxed area represents 50% of samples (from the 25 th to the 75 th percentile) and the band inside the box represents the median. Error bars (whiskers) indicate the 10th and 90th percentiles (shown only for plots with N = 9 or more). Symbols outside whiskers represent outliers.
    Figure Legend Snippet: The Iqgap2 promoter is not hypermethylated in hepatocellular carcinoma . The degree of methylation for each CpG site was expressed as a percentage of methylated cytosines over the sum of total cytosines. The data are shown for 25 CpG sites and grouped by tissue type. Genomic DNA from the Kato III human gastric cancer cell line, which is known to have high methylation levels of the Iqgap2 gene promoter [ 23 ], was used as a positive control. The boxed area represents 50% of samples (from the 25 th to the 75 th percentile) and the band inside the box represents the median. Error bars (whiskers) indicate the 10th and 90th percentiles (shown only for plots with N = 9 or more). Symbols outside whiskers represent outliers.

    Techniques Used: Methylation, Positive Control

    72) Product Images from "Sequential decitabine and carboplatin treatment increases the DNA repair protein XPC, increases apoptosis and decreases proliferation in melanoma"

    Article Title: Sequential decitabine and carboplatin treatment increases the DNA repair protein XPC, increases apoptosis and decreases proliferation in melanoma

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4010-9

    DNA methylation pattern of the XPC CpG island before and after decitabine. Methylation levels in each melanoma cell line at baseline (black) and after treatment with 0.26 μM decitabine (grey) was quantified by bisulfite sequencing. CpG position is shown relative to XPC transcription start site (TSS). Upstream (5′) shore = position − 2341 to − 423, CpG island = position − 364 to 568, Downstream (3′) shore = position 714 to 2596
    Figure Legend Snippet: DNA methylation pattern of the XPC CpG island before and after decitabine. Methylation levels in each melanoma cell line at baseline (black) and after treatment with 0.26 μM decitabine (grey) was quantified by bisulfite sequencing. CpG position is shown relative to XPC transcription start site (TSS). Upstream (5′) shore = position − 2341 to − 423, CpG island = position − 364 to 568, Downstream (3′) shore = position 714 to 2596

    Techniques Used: DNA Methylation Assay, Methylation, Methylation Sequencing

    73) Product Images from "Mitochondrial peptides modulate mitochondrial function during cellular senescence"

    Article Title: Mitochondrial peptides modulate mitochondrial function during cellular senescence

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101463

    Mitochondria DNA methylation changes during doxorubicin-induced senescence. ( A ) Schematic diagram of mitochondrial genes and CpG sites in the mtDNA. Quantification and representative agarose gel images of mtDNA methylation levels at the site of ( B ) CpG1 and ( C ) CpG4. ( D ) Quantification and representative western blots of COX1 (MT-CO1) in non-senescent and senescent cells. Reduced lamin B1 was used as a senescence marker. Data are reported as mean ± SEM of three to four independent experiments. Significant differences were determined by Student’s t -tests. *p
    Figure Legend Snippet: Mitochondria DNA methylation changes during doxorubicin-induced senescence. ( A ) Schematic diagram of mitochondrial genes and CpG sites in the mtDNA. Quantification and representative agarose gel images of mtDNA methylation levels at the site of ( B ) CpG1 and ( C ) CpG4. ( D ) Quantification and representative western blots of COX1 (MT-CO1) in non-senescent and senescent cells. Reduced lamin B1 was used as a senescence marker. Data are reported as mean ± SEM of three to four independent experiments. Significant differences were determined by Student’s t -tests. *p

    Techniques Used: DNA Methylation Assay, Agarose Gel Electrophoresis, Methylation, Western Blot, Marker

    Mitochondria mass and energetics are altered during doxorubicin-induced senescence. ( A ) mitochondrial DNA (mtDNA) copy number in non-senescent (quiescent) and senescent cells. ( B ) Representative images of Tom20 (green; mitochondria) and Hoechst 33258 (blue; nucleus) immunostaining in non-senescent (quiescent) and senescent cells. Scale bar, 20 μm. The area of Tom20 staining per cells were measured using image J. ( C ) Cellular ATP levels in non-senescent (quiescent) and senescent cells. ( D ) Cellular oxygen consumption rate (OCR) in non-senescent and senescent cells. The basal respiration, spare respiratory capacity, and ATP production are calculated based on the sequential compound injection according to the manufacture’s instruction. ( E ) The extracellular acidification rate (ECAR) in non-senescent (quiescent) and senescent cells. Glycolysis, glycolytic capacity, and glycolytic reserve are calculated based on the sequential compound injection according to the manufacture’s instruction. Data are reported as mean ± SEM of three to eight independent experiments. Significant differences were determined by Student’s t -tests. *p
    Figure Legend Snippet: Mitochondria mass and energetics are altered during doxorubicin-induced senescence. ( A ) mitochondrial DNA (mtDNA) copy number in non-senescent (quiescent) and senescent cells. ( B ) Representative images of Tom20 (green; mitochondria) and Hoechst 33258 (blue; nucleus) immunostaining in non-senescent (quiescent) and senescent cells. Scale bar, 20 μm. The area of Tom20 staining per cells were measured using image J. ( C ) Cellular ATP levels in non-senescent (quiescent) and senescent cells. ( D ) Cellular oxygen consumption rate (OCR) in non-senescent and senescent cells. The basal respiration, spare respiratory capacity, and ATP production are calculated based on the sequential compound injection according to the manufacture’s instruction. ( E ) The extracellular acidification rate (ECAR) in non-senescent (quiescent) and senescent cells. Glycolysis, glycolytic capacity, and glycolytic reserve are calculated based on the sequential compound injection according to the manufacture’s instruction. Data are reported as mean ± SEM of three to eight independent experiments. Significant differences were determined by Student’s t -tests. *p

    Techniques Used: Immunostaining, Staining, Injection

    74) Product Images from "Genome-Wide DNA Methylation Maps in Follicular Lymphoma Cells Determined by Methylation-Enriched Bisulfite Sequencing"

    Article Title: Genome-Wide DNA Methylation Maps in Follicular Lymphoma Cells Determined by Methylation-Enriched Bisulfite Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013020

    Experimental Design. A . Schematic diagram of MIRA-454 sequencing approach. B . Left panel: Gel electrophoresis of Mse I (M), Bfa I (B), and Csp 6I (C) digested genomic DNA. Right panel: PCR results of the adaptor-ligated and bisulfite-treated DNA. C . Histogram of the read length distribution of bisulfite sequences obtained from 454-sequencing of the methylation enriched genomic library. D . Mapping of the bisulfite sequencing reads along chromosome 7. A custom track was generated from alignment results of bisulfite sequences on human genome. The track was uploaded to the UCSC genome browser and compared with other tracks such as RefSeq genes and CpG islands.
    Figure Legend Snippet: Experimental Design. A . Schematic diagram of MIRA-454 sequencing approach. B . Left panel: Gel electrophoresis of Mse I (M), Bfa I (B), and Csp 6I (C) digested genomic DNA. Right panel: PCR results of the adaptor-ligated and bisulfite-treated DNA. C . Histogram of the read length distribution of bisulfite sequences obtained from 454-sequencing of the methylation enriched genomic library. D . Mapping of the bisulfite sequencing reads along chromosome 7. A custom track was generated from alignment results of bisulfite sequences on human genome. The track was uploaded to the UCSC genome browser and compared with other tracks such as RefSeq genes and CpG islands.

    Techniques Used: Sequencing, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Methylation, Methylation Sequencing, Generated

    75) Product Images from "The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice"

    Article Title: The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01003-12

    DNA methylation status of the ICR21 and ICR43 fragments in somatic cells of YAC-TgM. (A) Maps of the endogenous H19 , the ICR21, and the ICR43 fragments. Two regions (I and II) were analyzed by bisulfite sequencing and shown as gray bars above the transgene maps. (B and C) DNA methylation analysis of the ICR21 (B) and the ICR43 (C) regions in the TgM (lines 41 and 191) that inherited the transgenes either paternally (P) or maternally (M). Tail DNA was digested with XbaI and treated with sodium bisulfite. Region I of the ICR21 fragment (B) or region II of the ICR43 fragment (C) was amplified by nested PCR. PCR products were subcloned and then sequenced.
    Figure Legend Snippet: DNA methylation status of the ICR21 and ICR43 fragments in somatic cells of YAC-TgM. (A) Maps of the endogenous H19 , the ICR21, and the ICR43 fragments. Two regions (I and II) were analyzed by bisulfite sequencing and shown as gray bars above the transgene maps. (B and C) DNA methylation analysis of the ICR21 (B) and the ICR43 (C) regions in the TgM (lines 41 and 191) that inherited the transgenes either paternally (P) or maternally (M). Tail DNA was digested with XbaI and treated with sodium bisulfite. Region I of the ICR21 fragment (B) or region II of the ICR43 fragment (C) was amplified by nested PCR. PCR products were subcloned and then sequenced.

    Techniques Used: DNA Methylation Assay, Methylation Sequencing, Amplification, Nested PCR, Polymerase Chain Reaction

    DNA methylation status in the ICR43(λ+CTCF)21 YAC-TgM. (A) Map of the ICR43(λ+CTCF)21 fragment. Four regions (I, II′, III, and IV) were analyzed by bisulfite sequencing in panels B to E and shown as gray bars beneath the map. (B and C) DNA methylation statuses of the ICR43 (II′), the λ+CTCF (III and IV), and the ICR21 (I) regions in somatic cells of the ICR43(λ+CTCF)21 YAC-TgM (lines 2619 and 2653) that inherited the transgenes either maternally (B) or paternally (C). Tail DNA was digested with XbaI and treated with sodium bisulfite. The four regions were amplified by nested PCR. PCR products were subcloned and then sequenced. Each horizontal row represents a single DNA template molecule. Methylated (filled circles) and unmethylated (open circles) CpG motifs are shown. Gray bars indicate the locations of the CTCF binding sites. The overall percentage of methylated CpGs is indicated beside each panel. (D) DNA methylation statuses of the ICR43, the λ+CTCF, and the ICR21 regions in testis germ cells of the ICR43(λ+CTCF)21 YAC-TgM (lines 2619 and 2653). Genomic DNA from the testis was analyzed by bisulfite sequencing, as described for panels B and C. (E) DNA methylation status of the λ+CTCF (III) region in MII oocytes of the ICR43(λ+CTCF)21 YAC-TgM (lines 2619 and 2653). Oocytes were embedded in agarose beads and treated with sodium bisulfite. The beads were separately and directly used to amplify region III of the transgene by nested PCR, and the resulting fragments were individually subcloned and sequenced.
    Figure Legend Snippet: DNA methylation status in the ICR43(λ+CTCF)21 YAC-TgM. (A) Map of the ICR43(λ+CTCF)21 fragment. Four regions (I, II′, III, and IV) were analyzed by bisulfite sequencing in panels B to E and shown as gray bars beneath the map. (B and C) DNA methylation statuses of the ICR43 (II′), the λ+CTCF (III and IV), and the ICR21 (I) regions in somatic cells of the ICR43(λ+CTCF)21 YAC-TgM (lines 2619 and 2653) that inherited the transgenes either maternally (B) or paternally (C). Tail DNA was digested with XbaI and treated with sodium bisulfite. The four regions were amplified by nested PCR. PCR products were subcloned and then sequenced. Each horizontal row represents a single DNA template molecule. Methylated (filled circles) and unmethylated (open circles) CpG motifs are shown. Gray bars indicate the locations of the CTCF binding sites. The overall percentage of methylated CpGs is indicated beside each panel. (D) DNA methylation statuses of the ICR43, the λ+CTCF, and the ICR21 regions in testis germ cells of the ICR43(λ+CTCF)21 YAC-TgM (lines 2619 and 2653). Genomic DNA from the testis was analyzed by bisulfite sequencing, as described for panels B and C. (E) DNA methylation status of the λ+CTCF (III) region in MII oocytes of the ICR43(λ+CTCF)21 YAC-TgM (lines 2619 and 2653). Oocytes were embedded in agarose beads and treated with sodium bisulfite. The beads were separately and directly used to amplify region III of the transgene by nested PCR, and the resulting fragments were individually subcloned and sequenced.

    Techniques Used: DNA Methylation Assay, Methylation Sequencing, Amplification, Nested PCR, Polymerase Chain Reaction, Methylation, Binding Assay

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    Clone Assay:

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    Amplification:

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    SYBR Green Assay:

    Article Title: Epigenetic repression of Krüppel-like factor 4 through Dnmt1 contributes to EMT in renal fibrosis
    Article Snippet: Methylation-specific PCR (MSP) MSP was carried out on bisulfate-treated DNA using the EZ DNA Methylation-Gold™ kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. .. Bisulfite converted genomic DNA was PCR-amplified using methylation-specific primers and SYBR-Green reaction mix.

    Formalin-fixed Paraffin-Embedded:

    Article Title: DNA methylation profiling in the Carolina Breast Cancer Study defines cancer subclasses differing in clinicopathologic characteristics and survival
    Article Snippet: .. Bisulfite treatment of DNA Sodium bisulfite modification of DNA obtained from FFPE tissue was performed by using the EZ DNA Methylation Gold kit (Zymo Research, Orange, CA, USA) as previously described [ ]. .. Illumina GoldenGate Cancer Panel I methylation array analysis Array-based DNA methylation profiling was accomplished by using the Illumina GoldenGate Cancer Panel I methylation bead array to simultaneously interrogate 1505 CpG loci associated with 807 cancer-related genes.

    Article Title: Discovery and validation of DNA methylation markers for overall survival prognosis in patients with thymic epithelial tumors
    Article Snippet: Genomic DNA was extracted from 10 sections using the QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany). .. Bisulfite conversion of total 500 ng purified DNA in each sample was performed with EZ DNA Methylation-GoldTM Kit according to manufacturer’s instructions (Cat. No. D5006, Zymo Research Corporation, Orange, CA, USA).

    Expressing:

    Article Title: Supplementary outcomes of betaine on economic and productive performance, some biochemical parameters, and lipoprotein lipase gene expression in finishing male broilers
    Article Snippet: Paragraph title: LPL gene expression and promoter methylation assay: ... Briefly, bisulfite conversion was done using EZ DNA Gold Kit, D5005, D5006, (Zymo research, USA).

    Modification:

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: .. From each sample, 500 ng of genomic DNA was modified with sodium bisulfite (optimal range 200–500 ng) using the EZ DNA Methylation‐Gold™ Kit following the manufacturer's instructions (D5005, Zymo Research Corporation, Irvine, CA, USA). .. PCR was performed on the bisulfite‐converted DNA samples using the forward and reverse primers shown in Table .

    Article Title: DNA methylation profiling in the Carolina Breast Cancer Study defines cancer subclasses differing in clinicopathologic characteristics and survival
    Article Snippet: .. Bisulfite treatment of DNA Sodium bisulfite modification of DNA obtained from FFPE tissue was performed by using the EZ DNA Methylation Gold kit (Zymo Research, Orange, CA, USA) as previously described [ ]. .. Illumina GoldenGate Cancer Panel I methylation array analysis Array-based DNA methylation profiling was accomplished by using the Illumina GoldenGate Cancer Panel I methylation bead array to simultaneously interrogate 1505 CpG loci associated with 807 cancer-related genes.

    Article Title: Clinical significance of aberrant DEUP1 promoter methylation in hepatocellular carcinoma
    Article Snippet: .. The DNA was modified with sulfite using an EZ DNA Methylation-Gold™ kit D5005 (Zymo Research Corp., Irvine, CA, USA). .. The primers were designed using Primer Premier 5 (Premier Biosoft International).

    Hybridization:

    Article Title: Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring
    Article Snippet: Genomic DNA (200 ng) was bisulfite-converted using the EZ DNA methylation gold kit, (Zymo Research, Leiden, The Netherlands) according to the manufacturer's protocol. .. The PCR product was purified using streptavidin Sepharose HP beads and hybridization of the sequencing primer with the biotinylated PCR product was performed as described in the PyroMark Q24 vacuum workstation guide (Qiagen).

    Genomic Sequencing:

    Article Title: Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation
    Article Snippet: The extent of methylation at a specific site (expressed in %) was calculated from reference PCR reactions for genomic sequences lacking a GCGC tetranucleotide and from a parallel reaction of the same, but undigested, genomic DNA sample. .. Bisulfite conversion of fibroblast-derived DNA from two clones (S1sh_8 and S5sh_11) was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, CA, USA).

    Polymerase Chain Reaction:

    Article Title: Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation
    Article Snippet: The extent of methylation at a specific site (expressed in %) was calculated from reference PCR reactions for genomic sequences lacking a GCGC tetranucleotide and from a parallel reaction of the same, but undigested, genomic DNA sample. .. Bisulfite conversion of fibroblast-derived DNA from two clones (S1sh_8 and S5sh_11) was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, CA, USA).

    Article Title: Epigenetic repression of Krüppel-like factor 4 through Dnmt1 contributes to EMT in renal fibrosis
    Article Snippet: .. Methylation-specific PCR (MSP) MSP was carried out on bisulfate-treated DNA using the EZ DNA Methylation-Gold™ kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. .. Methylation-specific primers for the KLF4 promoter were designed using the MethPrimer program, as previously described ( ).

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: Forward/reverse and sequencing primers for the PCR and pyrosequencing steps, respectively, were designed from modified DNA sequences using the PyroMark Assay Design software version 2.0.1.15 (Qiagen, Uppsala, Sweden). .. From each sample, 500 ng of genomic DNA was modified with sodium bisulfite (optimal range 200–500 ng) using the EZ DNA Methylation‐Gold™ Kit following the manufacturer's instructions (D5005, Zymo Research Corporation, Irvine, CA, USA).

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: Bisulfite conversion of the DNA was carried out with EZ DNA Methylation‐Gold™ Kit D5005 (Zymo Research Corporation, Irvine, CA, USA). .. PCR products were cleaned with Exo‐SAP (Fermentas), sequenced with a BigDye V3.1 kit (#4336935, Applied Biosystems), and then purified using ethanol precipitation.

    Article Title: Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring
    Article Snippet: To identify common single-nucleotide polymorphisms that could interfere with the pyrosequencing assay itself, the PCR products were cloned and sequenced (Baseclear, Leiden, The Netherlands). .. Genomic DNA (200 ng) was bisulfite-converted using the EZ DNA methylation gold kit, (Zymo Research, Leiden, The Netherlands) according to the manufacturer's protocol.

    Article Title: Discovery and validation of DNA methylation markers for overall survival prognosis in patients with thymic epithelial tumors
    Article Snippet: Bisulfite conversion of total 500 ng purified DNA in each sample was performed with EZ DNA Methylation-GoldTM Kit according to manufacturer’s instructions (Cat. No. D5006, Zymo Research Corporation, Orange, CA, USA). .. The bisulfite conversed DNA was amplified with TaKaRa EpiTaqTM HS (Cat. No. R110A, Takara Biomedical Technology (Beijing) Co., Ltd. Beijing, China) with reaction setup: 10 ng bisulfite-treated DNA, 0.4 μM forward and reverse primers, 2.5 μL 10 × EpiTap PCR Buffer, 2.5 mM MgCl2, dNTP Mixture (0.264 mM each), EpiTap HS(0.025 U/μL) in total 25 μL each reaction and with following thermal cycle condition: denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s executed for 35 cycles followed by extension at 72 °C for 1 min and hold at 4 °C.

    Article Title: Nerve injury-induced epigenetic silencing of opioid receptors controlled by DNMT3a in primary afferent neurons
    Article Snippet: Bisulfite treatment of genomic DNA was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA) according to the manufacturer’s instructions. .. The master mix of the binding buffer, streptavidin-sepharose beads, and PCR products were prepared for the binding reaction in a 96-well plate.

    Binding Assay:

    Article Title: Nerve injury-induced epigenetic silencing of opioid receptors controlled by DNMT3a in primary afferent neurons
    Article Snippet: Bisulfite treatment of genomic DNA was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA) according to the manufacturer’s instructions. .. The master mix of the binding buffer, streptavidin-sepharose beads, and PCR products were prepared for the binding reaction in a 96-well plate.

    Pyromark Assay:

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: Forward/reverse and sequencing primers for the PCR and pyrosequencing steps, respectively, were designed from modified DNA sequences using the PyroMark Assay Design software version 2.0.1.15 (Qiagen, Uppsala, Sweden). .. From each sample, 500 ng of genomic DNA was modified with sodium bisulfite (optimal range 200–500 ng) using the EZ DNA Methylation‐Gold™ Kit following the manufacturer's instructions (D5005, Zymo Research Corporation, Irvine, CA, USA).

    Article Title: Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring
    Article Snippet: Bisulfite-specific primers were developed using the Pyromark Assay design 2.0 software (Qiagen). .. Genomic DNA (200 ng) was bisulfite-converted using the EZ DNA methylation gold kit, (Zymo Research, Leiden, The Netherlands) according to the manufacturer's protocol.

    DNA Extraction:

    Article Title: LINE-1 hypomethylation in normal colon mucosa is associated with poor survival in Chinese patients with sporadic colon cancer
    Article Snippet: Paragraph title: Genomic DNA isolation and bisulfite conversion ... Bisulfite treatment of 0.5–1 μg of gDNA (tumor or normal mucosa) was performed using methylation kits (#D5006, ZYMO Research, USA) according to the manufacturer's instructions.

    Article Title: Nerve injury-induced epigenetic silencing of opioid receptors controlled by DNMT3a in primary afferent neurons
    Article Snippet: Two DRGs were pooled together for DNA extraction. .. Bisulfite treatment of genomic DNA was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA) according to the manufacturer’s instructions.

    Methylation:

    Article Title: Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation
    Article Snippet: .. Bisulfite conversion of fibroblast-derived DNA from two clones (S1sh_8 and S5sh_11) was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, CA, USA). .. Primers flanking the methylation sites M1–4 in ICR1 were designed complementary to the bisulfite-converted sequence (SRSBSfor3, 5′-TTATGGGAATAGAGGGTTTG-′3, and SRSBSrev1, 5′-CCACTATCTCCCCTCAA-′3).

    Article Title: Epigenetic repression of Krüppel-like factor 4 through Dnmt1 contributes to EMT in renal fibrosis
    Article Snippet: .. Methylation-specific PCR (MSP) MSP was carried out on bisulfate-treated DNA using the EZ DNA Methylation-Gold™ kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. .. Methylation-specific primers for the KLF4 promoter were designed using the MethPrimer program, as previously described ( ).

    Article Title: LINE-1 hypomethylation in normal colon mucosa is associated with poor survival in Chinese patients with sporadic colon cancer
    Article Snippet: .. Bisulfite treatment of 0.5–1 μg of gDNA (tumor or normal mucosa) was performed using methylation kits (#D5006, ZYMO Research, USA) according to the manufacturer's instructions. .. Pyrosequencing for LINE-1 methylation levels Bisulfite-treated DNA samples from normal colon mucosa were subjected to PCR amplification using an ABI GeneAmp® PCR System 9700 (Applied Biosystems, USA); the 50-μL reactions contained 0.2 μL (5 U/μl) of KAPA Taq DNA Polymerase (Kapa Biosystems, USA), 50 pmol of each forward and reverse primer, and 2 μL of bisulfate-converted DNA.

    Article Title: Supplementary outcomes of betaine on economic and productive performance, some biochemical parameters, and lipoprotein lipase gene expression in finishing male broilers
    Article Snippet: Paragraph title: LPL gene expression and promoter methylation assay: ... Briefly, bisulfite conversion was done using EZ DNA Gold Kit, D5005, D5006, (Zymo research, USA).

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: .. From each sample, 500 ng of genomic DNA was modified with sodium bisulfite (optimal range 200–500 ng) using the EZ DNA Methylation‐Gold™ Kit following the manufacturer's instructions (D5005, Zymo Research Corporation, Irvine, CA, USA). .. PCR was performed on the bisulfite‐converted DNA samples using the forward and reverse primers shown in Table .

    Article Title: Clinical significance of aberrant DEUP1 promoter methylation in hepatocellular carcinoma
    Article Snippet: .. The DNA was modified with sulfite using an EZ DNA Methylation-Gold™ kit D5005 (Zymo Research Corp., Irvine, CA, USA). .. The primers were designed using Primer Premier 5 (Premier Biosoft International).

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: .. Bisulfite conversion of the DNA was carried out with EZ DNA Methylation‐Gold™ Kit D5005 (Zymo Research Corporation, Irvine, CA, USA). .. Primers for sequencing were designed with MethPrimer ( http://urogene.org/methprimer/index.html ).

    Article Title: Discovery and validation of DNA methylation markers for overall survival prognosis in patients with thymic epithelial tumors
    Article Snippet: .. Bisulfite conversion of total 500 ng purified DNA in each sample was performed with EZ DNA Methylation-GoldTM Kit according to manufacturer’s instructions (Cat. No. D5006, Zymo Research Corporation, Orange, CA, USA). .. The bisulfite conversed DNA was amplified with TaKaRa EpiTaqTM HS (Cat. No. R110A, Takara Biomedical Technology (Beijing) Co., Ltd. Beijing, China) with reaction setup: 10 ng bisulfite-treated DNA, 0.4 μM forward and reverse primers, 2.5 μL 10 × EpiTap PCR Buffer, 2.5 mM MgCl2, dNTP Mixture (0.264 mM each), EpiTap HS(0.025 U/μL) in total 25 μL each reaction and with following thermal cycle condition: denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s executed for 35 cycles followed by extension at 72 °C for 1 min and hold at 4 °C.

    Article Title: Nerve injury-induced epigenetic silencing of opioid receptors controlled by DNMT3a in primary afferent neurons
    Article Snippet: .. Bisulfite treatment of genomic DNA was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA) according to the manufacturer’s instructions. .. The region of the Oprm1 gene promoter from −670 to +450 bp consisted of 38 CpG sites was amplified.

    Isolation:

    Article Title: LINE-1 hypomethylation in normal colon mucosa is associated with poor survival in Chinese patients with sporadic colon cancer
    Article Snippet: Genomic DNA isolation and bisulfite conversion Genomic DNA (gDNA) was isolated from tumor or normal colonic mucosa tissue samples using tissue DNA isolation kits (#D3051, ZYMO Research, USA) according to the manufacturer's instructions. gDNA was quantified using a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific Inc., USA). .. Bisulfite treatment of 0.5–1 μg of gDNA (tumor or normal mucosa) was performed using methylation kits (#D5006, ZYMO Research, USA) according to the manufacturer's instructions.

    Purification:

    Article Title: Supplementary outcomes of betaine on economic and productive performance, some biochemical parameters, and lipoprotein lipase gene expression in finishing male broilers
    Article Snippet: 2.2 LPL gene expression and promoter methylation assay: Genomic DNA was purified from abdominal adipose tissues using DNasy tissue kit (QIAGEN, GmbH, Hilden, Germany) according to manufacturer instructions. .. Briefly, bisulfite conversion was done using EZ DNA Gold Kit, D5005, D5006, (Zymo research, USA).

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: Bisulfite conversion of the DNA was carried out with EZ DNA Methylation‐Gold™ Kit D5005 (Zymo Research Corporation, Irvine, CA, USA). .. PCR products were cleaned with Exo‐SAP (Fermentas), sequenced with a BigDye V3.1 kit (#4336935, Applied Biosystems), and then purified using ethanol precipitation.

    Article Title: Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring
    Article Snippet: Genomic DNA (200 ng) was bisulfite-converted using the EZ DNA methylation gold kit, (Zymo Research, Leiden, The Netherlands) according to the manufacturer's protocol. .. The PCR product was purified using streptavidin Sepharose HP beads and hybridization of the sequencing primer with the biotinylated PCR product was performed as described in the PyroMark Q24 vacuum workstation guide (Qiagen).

    Article Title: Discovery and validation of DNA methylation markers for overall survival prognosis in patients with thymic epithelial tumors
    Article Snippet: .. Bisulfite conversion of total 500 ng purified DNA in each sample was performed with EZ DNA Methylation-GoldTM Kit according to manufacturer’s instructions (Cat. No. D5006, Zymo Research Corporation, Orange, CA, USA). .. The bisulfite conversed DNA was amplified with TaKaRa EpiTaqTM HS (Cat. No. R110A, Takara Biomedical Technology (Beijing) Co., Ltd. Beijing, China) with reaction setup: 10 ng bisulfite-treated DNA, 0.4 μM forward and reverse primers, 2.5 μL 10 × EpiTap PCR Buffer, 2.5 mM MgCl2, dNTP Mixture (0.264 mM each), EpiTap HS(0.025 U/μL) in total 25 μL each reaction and with following thermal cycle condition: denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s executed for 35 cycles followed by extension at 72 °C for 1 min and hold at 4 °C.

    Sequencing:

    Article Title: Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation
    Article Snippet: Bisulfite conversion of fibroblast-derived DNA from two clones (S1sh_8 and S5sh_11) was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, CA, USA). .. Primers flanking the methylation sites M1–4 in ICR1 were designed complementary to the bisulfite-converted sequence (SRSBSfor3, 5′-TTATGGGAATAGAGGGTTTG-′3, and SRSBSrev1, 5′-CCACTATCTCCCCTCAA-′3).

    Article Title: Supplementary outcomes of betaine on economic and productive performance, some biochemical parameters, and lipoprotein lipase gene expression in finishing male broilers
    Article Snippet: Briefly, bisulfite conversion was done using EZ DNA Gold Kit, D5005, D5006, (Zymo research, USA). .. At each CpG site, the CpG of genomic DNA template was considered as methylated when the sequence obtained was TpG.

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: Forward/reverse and sequencing primers for the PCR and pyrosequencing steps, respectively, were designed from modified DNA sequences using the PyroMark Assay Design software version 2.0.1.15 (Qiagen, Uppsala, Sweden). .. From each sample, 500 ng of genomic DNA was modified with sodium bisulfite (optimal range 200–500 ng) using the EZ DNA Methylation‐Gold™ Kit following the manufacturer's instructions (D5005, Zymo Research Corporation, Irvine, CA, USA).

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: Bisulfite conversion of the DNA was carried out with EZ DNA Methylation‐Gold™ Kit D5005 (Zymo Research Corporation, Irvine, CA, USA). .. Primers for sequencing were designed with MethPrimer ( http://urogene.org/methprimer/index.html ).

    Article Title: Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring
    Article Snippet: From these sequence data, an A/T SNP was identified that could lead to out-of-frame sequencing; this was added as a non-CpG SNP to the sequence to analyze (AQ-mode). .. Genomic DNA (200 ng) was bisulfite-converted using the EZ DNA methylation gold kit, (Zymo Research, Leiden, The Netherlands) according to the manufacturer's protocol.

    Software:

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: Forward/reverse and sequencing primers for the PCR and pyrosequencing steps, respectively, were designed from modified DNA sequences using the PyroMark Assay Design software version 2.0.1.15 (Qiagen, Uppsala, Sweden). .. From each sample, 500 ng of genomic DNA was modified with sodium bisulfite (optimal range 200–500 ng) using the EZ DNA Methylation‐Gold™ Kit following the manufacturer's instructions (D5005, Zymo Research Corporation, Irvine, CA, USA).

    Article Title: Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring
    Article Snippet: Bisulfite-specific primers were developed using the Pyromark Assay design 2.0 software (Qiagen). .. Genomic DNA (200 ng) was bisulfite-converted using the EZ DNA methylation gold kit, (Zymo Research, Leiden, The Netherlands) according to the manufacturer's protocol.

    Real-time Polymerase Chain Reaction:

    Article Title: Supplementary outcomes of betaine on economic and productive performance, some biochemical parameters, and lipoprotein lipase gene expression in finishing male broilers
    Article Snippet: Primers obtained from different exons were used for both quantitative real time PCR (qrtPCR) and methylation of LPL gene promoter according to Xing et al. . .. Briefly, bisulfite conversion was done using EZ DNA Gold Kit, D5005, D5006, (Zymo research, USA).

    Agarose Gel Electrophoresis:

    Article Title: Clinical significance of aberrant DEUP1 promoter methylation in hepatocellular carcinoma
    Article Snippet: DEUP1 promoter methylation detected by BSP DNA in the tissues was extracted using a TINamp Genomic DNA kit (Sangon Biotech, Co., Ltd.) and resolved via 1% agarose gel electrophoresis. .. The DNA was modified with sulfite using an EZ DNA Methylation-Gold™ kit D5005 (Zymo Research Corp., Irvine, CA, USA).

    Ethanol Precipitation:

    Article Title: Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird. Age‐ and quality‐dependent DNA methylation correlate with melanin‐based coloration in a wild bird
    Article Snippet: Bisulfite conversion of the DNA was carried out with EZ DNA Methylation‐Gold™ Kit D5005 (Zymo Research Corporation, Irvine, CA, USA). .. PCR products were cleaned with Exo‐SAP (Fermentas), sequenced with a BigDye V3.1 kit (#4336935, Applied Biosystems), and then purified using ethanol precipitation.

    Spectrophotometry:

    Article Title: LINE-1 hypomethylation in normal colon mucosa is associated with poor survival in Chinese patients with sporadic colon cancer
    Article Snippet: Genomic DNA isolation and bisulfite conversion Genomic DNA (gDNA) was isolated from tumor or normal colonic mucosa tissue samples using tissue DNA isolation kits (#D3051, ZYMO Research, USA) according to the manufacturer's instructions. gDNA was quantified using a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific Inc., USA). .. Bisulfite treatment of 0.5–1 μg of gDNA (tumor or normal mucosa) was performed using methylation kits (#D5006, ZYMO Research, USA) according to the manufacturer's instructions.

    Article Title: Clinical significance of aberrant DEUP1 promoter methylation in hepatocellular carcinoma
    Article Snippet: The absorbance (260/280) was measured with a UV spectrophotometer to calculate DNA content. .. The DNA was modified with sulfite using an EZ DNA Methylation-Gold™ kit D5005 (Zymo Research Corp., Irvine, CA, USA).

    DNA Methylation Assay:

    Article Title: DNA methylation profiling in the Carolina Breast Cancer Study defines cancer subclasses differing in clinicopathologic characteristics and survival
    Article Snippet: .. Bisulfite treatment of DNA Sodium bisulfite modification of DNA obtained from FFPE tissue was performed by using the EZ DNA Methylation Gold kit (Zymo Research, Orange, CA, USA) as previously described [ ]. .. Illumina GoldenGate Cancer Panel I methylation array analysis Array-based DNA methylation profiling was accomplished by using the Illumina GoldenGate Cancer Panel I methylation bead array to simultaneously interrogate 1505 CpG loci associated with 807 cancer-related genes.

    Article Title: Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring
    Article Snippet: .. Genomic DNA (200 ng) was bisulfite-converted using the EZ DNA methylation gold kit, (Zymo Research, Leiden, The Netherlands) according to the manufacturer's protocol. .. LINE-1 was amplified by HotStar Mastermix (Qiagen) (see for the PCR conditions).

    Pyrosequencing Assay:

    Article Title: Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring
    Article Snippet: To identify common single-nucleotide polymorphisms that could interfere with the pyrosequencing assay itself, the PCR products were cloned and sequenced (Baseclear, Leiden, The Netherlands). .. Genomic DNA (200 ng) was bisulfite-converted using the EZ DNA methylation gold kit, (Zymo Research, Leiden, The Netherlands) according to the manufacturer's protocol.

    Article Title: Nerve injury-induced epigenetic silencing of opioid receptors controlled by DNMT3a in primary afferent neurons
    Article Snippet: Bisulfite treatment of genomic DNA was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA) according to the manufacturer’s instructions. .. For the pyrosequencing assay, 3 pairs of primers of the Oprm1 gene with 5′-biotin listed in the were used to amplifying the bisulfite DNA.

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    Zymo Research ez dna methylation gold kit
    <t>DNA</t> Methylation Differences Observed in MLL-AF4 + B Cell Blasts Versus CD34 + Cells Expressing MLL-AF4 and CD34 + CD19 + B Cell HPCs (A) Global DNA methylation analysis by <t>pyrosequencing</t> of LINE-1 elements in MLL-AF4 + blasts and normal CD34 + CD19 + B cell HPCs (n = 3 independent experiments). Error bars indicate SD. (B) Unsupervised hierarchical clustering and heatmap showing the CpG sites with differential DNA methylation between MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + cells expressing ectopic MLL-AF4. Average methylation values are displayed from 0 (blue) to 1 (yellow). (C) Venn diagrams showing the number of CpG sites differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. (D) Selection of GO terms from the top 50 statistically significant biological functions, ranked by p value (x axis), of genes differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. The y axis indicates the relative risk (±95% confidence intervals) as a measure of effect size. The relative risk is the ratio of the proportion of genes belonging to a given GO term in a selected subset of genes to the same proportion in the remaining, background genes.
    Ez Dna Methylation Gold Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 95/100, based on 1946 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA Methylation Differences Observed in MLL-AF4 + B Cell Blasts Versus CD34 + Cells Expressing MLL-AF4 and CD34 + CD19 + B Cell HPCs (A) Global DNA methylation analysis by pyrosequencing of LINE-1 elements in MLL-AF4 + blasts and normal CD34 + CD19 + B cell HPCs (n = 3 independent experiments). Error bars indicate SD. (B) Unsupervised hierarchical clustering and heatmap showing the CpG sites with differential DNA methylation between MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + cells expressing ectopic MLL-AF4. Average methylation values are displayed from 0 (blue) to 1 (yellow). (C) Venn diagrams showing the number of CpG sites differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. (D) Selection of GO terms from the top 50 statistically significant biological functions, ranked by p value (x axis), of genes differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. The y axis indicates the relative risk (±95% confidence intervals) as a measure of effect size. The relative risk is the ratio of the proportion of genes belonging to a given GO term in a selected subset of genes to the same proportion in the remaining, background genes.

    Journal: Stem Cell Reports

    Article Title: Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

    doi: 10.1016/j.stemcr.2016.08.013

    Figure Lengend Snippet: DNA Methylation Differences Observed in MLL-AF4 + B Cell Blasts Versus CD34 + Cells Expressing MLL-AF4 and CD34 + CD19 + B Cell HPCs (A) Global DNA methylation analysis by pyrosequencing of LINE-1 elements in MLL-AF4 + blasts and normal CD34 + CD19 + B cell HPCs (n = 3 independent experiments). Error bars indicate SD. (B) Unsupervised hierarchical clustering and heatmap showing the CpG sites with differential DNA methylation between MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + cells expressing ectopic MLL-AF4. Average methylation values are displayed from 0 (blue) to 1 (yellow). (C) Venn diagrams showing the number of CpG sites differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. (D) Selection of GO terms from the top 50 statistically significant biological functions, ranked by p value (x axis), of genes differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. The y axis indicates the relative risk (±95% confidence intervals) as a measure of effect size. The relative risk is the ratio of the proportion of genes belonging to a given GO term in a selected subset of genes to the same proportion in the remaining, background genes.

    Article Snippet: Bisulfite Pyrosequencing, Human Methylation 450 BeadChip Array, and Data Analysis Differences in global DNA methylation were compared between MLL-AF4+ blasts and healthy CD34+ CD19+ B cell HPCs and in B cell lines before and after decitabine treatment by bisulfite pyrosequencing of LINE-1 elements using the EZ DNA Methylation-Gold kit (Zymo Research).

    Techniques: DNA Methylation Assay, Expressing, Methylation, Selection

    Tethered SUVH2 recruits Pol V through DRD1, resulting in DNA methylation and a late-flowering phenotype a. Plants grown side-by-side to illustrate early flowering of ZF-SUVH2 in fwa-4 (T2 plants) compared to fwa-4. b. Flowering time of Columbia-0 (WT), ZF-SUVH2 in fwa-4 , ZF-KYP in fwa-4 , HA-SUVH2 in fwa-4 , and fwa-4 . Flowering time was determined by counting all rosette and cauline leaves up until the terminal flower. The average leaf number and standard deviation of between 20-30 plants was determined. Mean +/− SD. c. Percent methylation at each cytosine in the FWA repeat region as determined by BS-seq in T2 and T3 ZF-SUVH2/ fwa-4 plants compared to T2 ZF-KYP/ fwa-4 (unmethylated) and WT (standard methylation pattern). ZF binding sites are shown in green and the FWA gene in blue. d. NRPE1 ChIP in WT (positive control), nrpe1 mutant (negative control), fwa-4 epiallele, and ZF-SUVH2/ fwa-4 . qPCR results of two well-characterized NRPE1 binding sites (IGN5 and IGN22) and two regions in FWA (FWAp: promoter; FWAt: transcript) are shown as enrichment of IP/input relative to negative control. Mean +/− SD of two biological replicas. e. Coimmunoprecipitation of HA-SUVH2 in Arabidopsis using Flag-DRD1. Left panels are inputs from the two parental strains (expressing either HA-SUVH2 (HA-2) or Flag-DRD1 (Flag-D)) and an F2 line expressing both HA-SUVH2 and Flag-DRD1 (HA-2xFlag-d). The right panels show elution from Flag-magnetic beads. Top panels are HA western blots, bottom panels are Flag western blots.

    Journal: Nature

    Article Title: SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation

    doi: 10.1038/nature12931

    Figure Lengend Snippet: Tethered SUVH2 recruits Pol V through DRD1, resulting in DNA methylation and a late-flowering phenotype a. Plants grown side-by-side to illustrate early flowering of ZF-SUVH2 in fwa-4 (T2 plants) compared to fwa-4. b. Flowering time of Columbia-0 (WT), ZF-SUVH2 in fwa-4 , ZF-KYP in fwa-4 , HA-SUVH2 in fwa-4 , and fwa-4 . Flowering time was determined by counting all rosette and cauline leaves up until the terminal flower. The average leaf number and standard deviation of between 20-30 plants was determined. Mean +/− SD. c. Percent methylation at each cytosine in the FWA repeat region as determined by BS-seq in T2 and T3 ZF-SUVH2/ fwa-4 plants compared to T2 ZF-KYP/ fwa-4 (unmethylated) and WT (standard methylation pattern). ZF binding sites are shown in green and the FWA gene in blue. d. NRPE1 ChIP in WT (positive control), nrpe1 mutant (negative control), fwa-4 epiallele, and ZF-SUVH2/ fwa-4 . qPCR results of two well-characterized NRPE1 binding sites (IGN5 and IGN22) and two regions in FWA (FWAp: promoter; FWAt: transcript) are shown as enrichment of IP/input relative to negative control. Mean +/− SD of two biological replicas. e. Coimmunoprecipitation of HA-SUVH2 in Arabidopsis using Flag-DRD1. Left panels are inputs from the two parental strains (expressing either HA-SUVH2 (HA-2) or Flag-DRD1 (Flag-D)) and an F2 line expressing both HA-SUVH2 and Flag-DRD1 (HA-2xFlag-d). The right panels show elution from Flag-magnetic beads. Top panels are HA western blots, bottom panels are Flag western blots.

    Article Snippet: Bisulfite sequencing followed by PCR amplification and cloning of FWA fragments was done using EZ DNA Methylation-Gold kit (Zymo Research) as described previously .

    Techniques: DNA Methylation Assay, Standard Deviation, Methylation, Binding Assay, Chromatin Immunoprecipitation, Positive Control, Mutagenesis, Negative Control, Real-time Polymerase Chain Reaction, Expressing, Magnetic Beads, Western Blot

    ZF-SUVH2 construct stably recruits Pol V to FWA through a direct interaction with DRD1 a. Top: Diagram of SUVH2 with Zn Finger (ZF) inserted immediately before the HA tag. Bottom: Schematic of FWA gene showing the two small and two large repeats (blue arrows), the regions amplified by PCR (promoter and transcript: green lines), the start and direction of transcription (red arrow), and the sites bound by the ZF (indicated by two orange arrows). b. Flag-ChIP in WT versus ZF-KYP (flag-tagged) showing enrichment at FWA in both the promoter and transcript region (see above). c. Percent methylation at each C in the FWA repeat region from three individual T1 plants. Percent methylation was determined from 20-25 clones of bisulfite-treated DNA. d. BS-Seq of FWA from a Basta-resistant line containing the ZF-SUVH2 transgene and two Basta-sensitivie siblings which had lost the ZF-SUVH2 transgene. e. Pull-down of DRD1-Flag with ZF-SUVH2. Input: DRD1-Flag extract from Arabidopsis; Beads-mock: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana extract; Beads-ZF-SUVH2: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana ZF-SUVH2 extract. Top panel: Flag blot; bottom panel: HA blot.

    Journal: Nature

    Article Title: SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation

    doi: 10.1038/nature12931

    Figure Lengend Snippet: ZF-SUVH2 construct stably recruits Pol V to FWA through a direct interaction with DRD1 a. Top: Diagram of SUVH2 with Zn Finger (ZF) inserted immediately before the HA tag. Bottom: Schematic of FWA gene showing the two small and two large repeats (blue arrows), the regions amplified by PCR (promoter and transcript: green lines), the start and direction of transcription (red arrow), and the sites bound by the ZF (indicated by two orange arrows). b. Flag-ChIP in WT versus ZF-KYP (flag-tagged) showing enrichment at FWA in both the promoter and transcript region (see above). c. Percent methylation at each C in the FWA repeat region from three individual T1 plants. Percent methylation was determined from 20-25 clones of bisulfite-treated DNA. d. BS-Seq of FWA from a Basta-resistant line containing the ZF-SUVH2 transgene and two Basta-sensitivie siblings which had lost the ZF-SUVH2 transgene. e. Pull-down of DRD1-Flag with ZF-SUVH2. Input: DRD1-Flag extract from Arabidopsis; Beads-mock: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana extract; Beads-ZF-SUVH2: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana ZF-SUVH2 extract. Top panel: Flag blot; bottom panel: HA blot.

    Article Snippet: Bisulfite sequencing followed by PCR amplification and cloning of FWA fragments was done using EZ DNA Methylation-Gold kit (Zymo Research) as described previously .

    Techniques: Construct, Stable Transfection, Amplification, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Methylation, Clone Assay, Magnetic Beads

    Validation of top-ranked DMP cg20475486 using a clinical biomarker assay. Replication of the top-ranked DMP associated with aGVHD severity, cg20475486, using a semi-quantitative DNA methylation assay. a Box-and-whisker plot of DNA methylation values in graft donors in T cell-depleted HSCT (initial discovery cohort). We replicated the DNA hypomethylation phenotype in HSCT donors matched to recipients with severe aGVHD compared to no/mild aGVHD ( P = 0.039, Wilcoxon rank-sum test). b At a relative DNA methylation threshold of 8.295 (dotted line), the AUC was 0.74 with a maximal specificity and sensitivity of 0.75 and 0.71, respectively. c Box-and-whisker plot of DNA methylation values in graft donors in T cell-replete HSCT (that is, without the application of in vivo alemtuzumab). In an independent sample cohort, we confirmed the observed DNA methylation phenotype, suggesting the epigenetic classifier is also effective in the context of a T cell-replete conditioning regimen ( P = 0.050). For two samples, C t -values could not be detected in the MethyLight experiments. d At a threshold of PMR = 17.73 (dotted line), the area under the ROC curve was 0.73 with a maximal specificity and sensitivity of 0.71 and 0.78, respectively

    Journal: Genome Medicine

    Article Title: A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation

    doi: 10.1186/s13073-015-0246-z

    Figure Lengend Snippet: Validation of top-ranked DMP cg20475486 using a clinical biomarker assay. Replication of the top-ranked DMP associated with aGVHD severity, cg20475486, using a semi-quantitative DNA methylation assay. a Box-and-whisker plot of DNA methylation values in graft donors in T cell-depleted HSCT (initial discovery cohort). We replicated the DNA hypomethylation phenotype in HSCT donors matched to recipients with severe aGVHD compared to no/mild aGVHD ( P = 0.039, Wilcoxon rank-sum test). b At a relative DNA methylation threshold of 8.295 (dotted line), the AUC was 0.74 with a maximal specificity and sensitivity of 0.75 and 0.71, respectively. c Box-and-whisker plot of DNA methylation values in graft donors in T cell-replete HSCT (that is, without the application of in vivo alemtuzumab). In an independent sample cohort, we confirmed the observed DNA methylation phenotype, suggesting the epigenetic classifier is also effective in the context of a T cell-replete conditioning regimen ( P = 0.050). For two samples, C t -values could not be detected in the MethyLight experiments. d At a threshold of PMR = 17.73 (dotted line), the area under the ROC curve was 0.73 with a maximal specificity and sensitivity of 0.71 and 0.78, respectively

    Article Snippet: Measurement of relative DNA methylation levels using MethyLight Genomic DNA was bisulfite-converted using an EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s instructions.

    Techniques: Biomarker Assay, DNA Methylation Assay, Whisker Assay, In Vivo

    Overview of the study design. We aimed to identify specific epigenetic marks in peripheral blood of healthy graft donors that delineate aGVHD severity in HLA-matched sibling recipients prior to HSCT. At the discovery stage, we assessed genome-wide DNA methylation levels in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, that is, ‘severe’ aGVHD (grades III + IV; n = 9) and ‘no/mild’ aGVHD (grades 0, I + II; n = 76). HSCT recipients received reduced-intensity (non-myeloablative) T cell-depleted conditioning using in vivo alemtuzumab. At the replication stage, we used a semi-quantitative DNA methylation assay, MethyLight, which can be easily used in a clinical setting. We validated the top-ranked differentially methylated positions associated with aGVHD severity status in donors in the context of both T cell-depleted and T cell-replete conditioning regimens for HSCT

    Journal: Genome Medicine

    Article Title: A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation

    doi: 10.1186/s13073-015-0246-z

    Figure Lengend Snippet: Overview of the study design. We aimed to identify specific epigenetic marks in peripheral blood of healthy graft donors that delineate aGVHD severity in HLA-matched sibling recipients prior to HSCT. At the discovery stage, we assessed genome-wide DNA methylation levels in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, that is, ‘severe’ aGVHD (grades III + IV; n = 9) and ‘no/mild’ aGVHD (grades 0, I + II; n = 76). HSCT recipients received reduced-intensity (non-myeloablative) T cell-depleted conditioning using in vivo alemtuzumab. At the replication stage, we used a semi-quantitative DNA methylation assay, MethyLight, which can be easily used in a clinical setting. We validated the top-ranked differentially methylated positions associated with aGVHD severity status in donors in the context of both T cell-depleted and T cell-replete conditioning regimens for HSCT

    Article Snippet: Measurement of relative DNA methylation levels using MethyLight Genomic DNA was bisulfite-converted using an EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s instructions.

    Techniques: Genome Wide, DNA Methylation Assay, In Vivo, Methylation

    Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P

    Journal: Journal of Neuroendocrinology

    Article Title: Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat

    doi: 10.1111/jne.12371

    Figure Lengend Snippet: Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P

    Article Snippet: Bisulphite conversion of DNA and TA cloning Genomic DNA from SON and cortex punches (50 ng) and rat hypothalamic 4B cells (200 ng) was bisulphite converted using EZ DNA Methylation‐Gold kit (Zymo Research).

    Techniques: Methylation, Clone Assay