input dna  (Millipore)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Name:
    Deoxyribonucleic acid
    Description:
    Human placental DNA is isolated from donor placenta but will contain some maternal DNA The DNA fragments are sonicated to produce fragments of consistent size
    Catalog Number:
    D3287
    Price:
    None
    Applications:
    Sonicated Deoxyribonucleic acid, single stranded from human placenta, was used as blocking agent in Southern hybridization of DNA from human papillomavirus (HPV) positive SiHa, HeLa and CaSki cell-lines. It was used as standard in GC/MS analysis of exocyclic DNA adducts.
    Buy from Supplier


    Structured Review

    Millipore input dna
    Binding of NF-κB and p53 on the endogenous galectin-7 promoter. Binding of NF-κB <t>p50</t> and c-Rel isoform, p53 and RNA polymerase II (PolII) on the endogenous galectin-7 promoter was measured by ChIP assay using genomic <t>DNA</t> collected from HaCaT ( A ) and human breast cancer cell lines MDA-MB-468 ( B ) and MCF-7 ( C ). An isotypic control (IgG) was used as a negative control. Total DNA extract was used as a positive control (Input).
    Human placental DNA is isolated from donor placenta but will contain some maternal DNA The DNA fragments are sonicated to produce fragments of consistent size
    https://www.bioz.com/result/input dna/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    input dna - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53"

    Article Title: Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072468

    Binding of NF-κB and p53 on the endogenous galectin-7 promoter. Binding of NF-κB p50 and c-Rel isoform, p53 and RNA polymerase II (PolII) on the endogenous galectin-7 promoter was measured by ChIP assay using genomic DNA collected from HaCaT ( A ) and human breast cancer cell lines MDA-MB-468 ( B ) and MCF-7 ( C ). An isotypic control (IgG) was used as a negative control. Total DNA extract was used as a positive control (Input).
    Figure Legend Snippet: Binding of NF-κB and p53 on the endogenous galectin-7 promoter. Binding of NF-κB p50 and c-Rel isoform, p53 and RNA polymerase II (PolII) on the endogenous galectin-7 promoter was measured by ChIP assay using genomic DNA collected from HaCaT ( A ) and human breast cancer cell lines MDA-MB-468 ( B ) and MCF-7 ( C ). An isotypic control (IgG) was used as a negative control. Total DNA extract was used as a positive control (Input).

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Multiple Displacement Amplification, Negative Control, Positive Control

    2) Product Images from "Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore"

    Article Title: Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2010.329

    Active centromere chromatin displays the signature of elongating RNA polymerase. ( A ) Schematic of the HAC, derived from Nakano et al (2008) , indicating the synthetic alphoid tetO array (green: tetO; white: CENP-B box) and the HAC vector with YAC and BAC cassettes and the blasticidin (Bsr) resistance marker. The region of the alphoid tetO array analysed by ChIP is indicated by green line. ( B ) ChIP analysis in AB2.2.18.21 cells using antibodies of the indicated reactivity. The synthetic (alphoid tetO ) centromere, endogenous chromosome 21 α21-I satellite DNA (alphoid chr.21 ), the 5S rDNA loci and the Bsr gene on the HAC vector were assessed. Data represent the mean and s.d. of three independent ChIP experiments. Note the different scaling of individual panels reflecting different efficiencies of individual antibodies. ( C ) Real-time RT–PCR analysis of synthetic HAC centromere (alphoid tetO ), actively transcribed Bsr marker and endogenous chromosome 21 satellite (alphoid chr.21 ). Expression data are normalized to the copy number of the genomic regions and β-actin mRNA levels (see Materials and methods) and displayed as arbitrary numbers. Data represent the mean and s.e.m. of three independent experiments. Note the log scale.
    Figure Legend Snippet: Active centromere chromatin displays the signature of elongating RNA polymerase. ( A ) Schematic of the HAC, derived from Nakano et al (2008) , indicating the synthetic alphoid tetO array (green: tetO; white: CENP-B box) and the HAC vector with YAC and BAC cassettes and the blasticidin (Bsr) resistance marker. The region of the alphoid tetO array analysed by ChIP is indicated by green line. ( B ) ChIP analysis in AB2.2.18.21 cells using antibodies of the indicated reactivity. The synthetic (alphoid tetO ) centromere, endogenous chromosome 21 α21-I satellite DNA (alphoid chr.21 ), the 5S rDNA loci and the Bsr gene on the HAC vector were assessed. Data represent the mean and s.d. of three independent ChIP experiments. Note the different scaling of individual panels reflecting different efficiencies of individual antibodies. ( C ) Real-time RT–PCR analysis of synthetic HAC centromere (alphoid tetO ), actively transcribed Bsr marker and endogenous chromosome 21 satellite (alphoid chr.21 ). Expression data are normalized to the copy number of the genomic regions and β-actin mRNA levels (see Materials and methods) and displayed as arbitrary numbers. Data represent the mean and s.e.m. of three independent experiments. Note the log scale.

    Techniques Used: HAC Assay, Derivative Assay, Plasmid Preparation, BAC Assay, Marker, Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing

    3) Product Images from "Salmonella expresses foreign genes during infection by degrading their silencer"

    Article Title: Salmonella expresses foreign genes during infection by degrading their silencer

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

    doi: 10.1073/pnas.1912808117

    Salmonella expresses horizontally transferred genes by degrading their silencer, H-NS. HTGs are transcriptionally repressed by H-NS, which binds to their regulatory regions. ( Left ) Under conditions resulting in constant high amounts of H-NS (such as neutral pH conditions or outside macrophages), only foreign genes targeted by antisilencing protein(s) are expressed. H-NS still binds to HTGs not bound by antisilencing proteins and silences their expression. ( Right ) When Salmonella is inside an acidic macrophage phagosome, DNA-binding antisilencing proteins (e.g., PhoP, SsrB, and SlyA) bind to the promoters of HTGs and displace H-NS from DNA. Lon degrades displaced H-NS. The resulting decrease in H-NS amounts de-represses HTGs, even those not bound by antisilencing proteins.
    Figure Legend Snippet: Salmonella expresses horizontally transferred genes by degrading their silencer, H-NS. HTGs are transcriptionally repressed by H-NS, which binds to their regulatory regions. ( Left ) Under conditions resulting in constant high amounts of H-NS (such as neutral pH conditions or outside macrophages), only foreign genes targeted by antisilencing protein(s) are expressed. H-NS still binds to HTGs not bound by antisilencing proteins and silences their expression. ( Right ) When Salmonella is inside an acidic macrophage phagosome, DNA-binding antisilencing proteins (e.g., PhoP, SsrB, and SlyA) bind to the promoters of HTGs and displace H-NS from DNA. Lon degrades displaced H-NS. The resulting decrease in H-NS amounts de-represses HTGs, even those not bound by antisilencing proteins.

    Techniques Used: Expressing, Binding Assay

    PhoP displaces H-NS from DNA, rendering it susceptible to degradation by Lon. ( A ) In vitro degradation of purified H-NS protein by purified Lon protease in the presence or absence of a plasmid harboring a foreign DNA fragment containing PhoP-activated genes targeted by H-NS (pHTG) and (where indicated) purified wild-type PhoP or variant PhoP (V193M) proteins. Pyruvate kinase (PK) is part of the ATP generation mix. t 1/2 , the half-life of H-NS. A representative of two independent experiments is shown. ( Bottom ) A densitometry graph for replicates. ( B ) Schematic of H-NS and the H-NS 1−50aa -GFP fusion proteins. AA, amino acids. ( C ) Western blot analysis of crude extracts prepared from wild-type (JC805), phoP (JC837), and lon (JC864) Salmonella harboring plasmids expressing the H-NS 1−50aa -GFP fusion protein using antibodies recognizing GFP or the loading control AtpB. Bacteria were grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH) with 20 µM of IPTG. Numbers above the blots correspond to the normalized relative amounts of H-NS 1−50aa -GFP protein. A representative of at least three independent experiments is shown.
    Figure Legend Snippet: PhoP displaces H-NS from DNA, rendering it susceptible to degradation by Lon. ( A ) In vitro degradation of purified H-NS protein by purified Lon protease in the presence or absence of a plasmid harboring a foreign DNA fragment containing PhoP-activated genes targeted by H-NS (pHTG) and (where indicated) purified wild-type PhoP or variant PhoP (V193M) proteins. Pyruvate kinase (PK) is part of the ATP generation mix. t 1/2 , the half-life of H-NS. A representative of two independent experiments is shown. ( Bottom ) A densitometry graph for replicates. ( B ) Schematic of H-NS and the H-NS 1−50aa -GFP fusion proteins. AA, amino acids. ( C ) Western blot analysis of crude extracts prepared from wild-type (JC805), phoP (JC837), and lon (JC864) Salmonella harboring plasmids expressing the H-NS 1−50aa -GFP fusion protein using antibodies recognizing GFP or the loading control AtpB. Bacteria were grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH) with 20 µM of IPTG. Numbers above the blots correspond to the normalized relative amounts of H-NS 1−50aa -GFP protein. A representative of at least three independent experiments is shown.

    Techniques Used: In Vitro, Purification, Plasmid Preparation, Variant Assay, Western Blot, Expressing

    Degradation of H-NS by the Lon protease decreases H-NS binding to HTGs, promoting their expression. ( A and B ) Western blot analysis of crude extracts prepared from ( A ) wild-type (JC805), phoP (JC837), and lon (JC864) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH) or pH 7.6 (neutral pH) and ( B ) wild-type (JC805), lon (JC864), clpX (JC865), and clpA (JC867) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH) using antibodies recognizing the FLAG epitope or the loading control AtpB. Numbers above the blots correspond to the normalized relative amounts of H-NS protein. ( C ) Stability of H-NS was determined in wild-type (JC805), phoP (JC837), and lon (JC864) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH). Protein synthesis was inhibited with chloramphenicol (1 mg⋅ml −1 ). Samples were removed at the indicated times and analyzed by Western blotting using antibodies recognizing the FLAG epitope or the loading control GroEL. t 1/2 , half-life of H-NS. ( D ) Western blot analysis of crude extracts prepared from wild-type (JC805) and lon (JC864) Salmonella with or without plasmids expressing wild-type Lon or a variant defective in DNA binding (Lonmu; R306E K308E K310E K311E) using antibodies recognizing the FLAG epitope, Lon, or the loading control AtpB. Bacteria were grown in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH) to midlog phase. L-Arabinose was added at the denoted final concentrations. Numbers above the blots correspond to the normalized relative amounts of H-NS protein. Representatives of at least three independent experiments are shown ( A – D ). ( E ) mRNA abundance of the pagC , STM14_1977 , and ycjE genes produced by wild-type (JC805), phoP (JC837), and lon (JC864) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH). ( F ) In vivo binding of H-NS to the promoter regions of the pagC , STM14_1977 , and ycjE genes was determined in wild-type (JC805) and lon (JC864) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH). ( G ) In vivo binding of PhoP to the promoter regions of the pagC , STM14_1977 , and ycjE genes was determined in wild-type (JC805) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 7.6 (neutral pH) or pH 4.9 (acidic pH). The mean and SD from three independent experiments are shown. * P
    Figure Legend Snippet: Degradation of H-NS by the Lon protease decreases H-NS binding to HTGs, promoting their expression. ( A and B ) Western blot analysis of crude extracts prepared from ( A ) wild-type (JC805), phoP (JC837), and lon (JC864) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH) or pH 7.6 (neutral pH) and ( B ) wild-type (JC805), lon (JC864), clpX (JC865), and clpA (JC867) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH) using antibodies recognizing the FLAG epitope or the loading control AtpB. Numbers above the blots correspond to the normalized relative amounts of H-NS protein. ( C ) Stability of H-NS was determined in wild-type (JC805), phoP (JC837), and lon (JC864) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH). Protein synthesis was inhibited with chloramphenicol (1 mg⋅ml −1 ). Samples were removed at the indicated times and analyzed by Western blotting using antibodies recognizing the FLAG epitope or the loading control GroEL. t 1/2 , half-life of H-NS. ( D ) Western blot analysis of crude extracts prepared from wild-type (JC805) and lon (JC864) Salmonella with or without plasmids expressing wild-type Lon or a variant defective in DNA binding (Lonmu; R306E K308E K310E K311E) using antibodies recognizing the FLAG epitope, Lon, or the loading control AtpB. Bacteria were grown in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH) to midlog phase. L-Arabinose was added at the denoted final concentrations. Numbers above the blots correspond to the normalized relative amounts of H-NS protein. Representatives of at least three independent experiments are shown ( A – D ). ( E ) mRNA abundance of the pagC , STM14_1977 , and ycjE genes produced by wild-type (JC805), phoP (JC837), and lon (JC864) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH). ( F ) In vivo binding of H-NS to the promoter regions of the pagC , STM14_1977 , and ycjE genes was determined in wild-type (JC805) and lon (JC864) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 4.9 (acidic pH). ( G ) In vivo binding of PhoP to the promoter regions of the pagC , STM14_1977 , and ycjE genes was determined in wild-type (JC805) Salmonella grown to midlog phase in N-minimal media with 1 mM Mg 2+ at pH 7.6 (neutral pH) or pH 4.9 (acidic pH). The mean and SD from three independent experiments are shown. * P

    Techniques Used: Binding Assay, Expressing, Western Blot, FLAG-tag, Variant Assay, Produced, In Vivo

    4) Product Images from "Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome"

    Article Title: Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-016-0181-x

    FMR1 locus (h)MeDIP profiling identifies novel regions of 5mC and 5hmC changes in FXS patient PBMC samples. a . Experimental overview. Methylated DNA immunoprecipitation (MeDIP) assay was used to profile DNA methylation (5mC), DNA hydroxymethylation (5hmC). Chromatin immunoprecipitation (ChIP) was used to profile histone post-translational modifications (PTM). Antibody (Ab) b – c . The graphs illustrate the relative enrichment of the indicated mark (log2 fold enrichment) in FXS ( red ) and control ( blue ) PBMCs. The upper panel illustrates the chromosomal and genomic location locations as well as the indicated referenced Refseq genes: FMR1 , and referenced antisense non coding RNAs ( FMR1AS1 and L29074.3). This snapshot illustrates the epigenetic landscape over a region covering 79 kb on ChrX: 146971000-147050000 ( c ). RPL19 and GAPDH provide controls regions of no changes in 5mC and 5hmC ( b ). In the graphs, the thicker lines indicate higher deviation between biological replicates (controls n = 4; FXS n = 8) (aggregation mean, sliding window 529 bp). Regions I to V were selected as regions of strongest apparent epigenetic variation across epigenetic marks and cell types based on epigenomic landscape visualization illustrated in Figs. 1 and 3 . The chromosome coordinates of these regions are provided in Table 1
    Figure Legend Snippet: FMR1 locus (h)MeDIP profiling identifies novel regions of 5mC and 5hmC changes in FXS patient PBMC samples. a . Experimental overview. Methylated DNA immunoprecipitation (MeDIP) assay was used to profile DNA methylation (5mC), DNA hydroxymethylation (5hmC). Chromatin immunoprecipitation (ChIP) was used to profile histone post-translational modifications (PTM). Antibody (Ab) b – c . The graphs illustrate the relative enrichment of the indicated mark (log2 fold enrichment) in FXS ( red ) and control ( blue ) PBMCs. The upper panel illustrates the chromosomal and genomic location locations as well as the indicated referenced Refseq genes: FMR1 , and referenced antisense non coding RNAs ( FMR1AS1 and L29074.3). This snapshot illustrates the epigenetic landscape over a region covering 79 kb on ChrX: 146971000-147050000 ( c ). RPL19 and GAPDH provide controls regions of no changes in 5mC and 5hmC ( b ). In the graphs, the thicker lines indicate higher deviation between biological replicates (controls n = 4; FXS n = 8) (aggregation mean, sliding window 529 bp). Regions I to V were selected as regions of strongest apparent epigenetic variation across epigenetic marks and cell types based on epigenomic landscape visualization illustrated in Figs. 1 and 3 . The chromosome coordinates of these regions are provided in Table 1

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

    5) Product Images from "Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes"

    Article Title: Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes

    Journal: Nature Communications

    doi: 10.1038/ncomms3889

    Folate deficiency alters sperm DNA methylation. Changes in DNA methylation are illustrated by smoothed MeDIP over input log2 ratios of individual oligonucleotides for the folate-sufficient (FS) and the folate-deficient (FD) animals. The gene is indicated at the bottom of the graph and the arrow represents the transcription start site (TSS).
    Figure Legend Snippet: Folate deficiency alters sperm DNA methylation. Changes in DNA methylation are illustrated by smoothed MeDIP over input log2 ratios of individual oligonucleotides for the folate-sufficient (FS) and the folate-deficient (FD) animals. The gene is indicated at the bottom of the graph and the arrow represents the transcription start site (TSS).

    Techniques Used: DNA Methylation Assay, Methylated DNA Immunoprecipitation

    Folate deficiency alters sperm DNA methylation at genes implicated in development and metabolic processes. Sequenom MassARRAY methylation analysis was performed on selected targets of interest identified by MeDIP-chip as altered in FD versus FS sperm. FD sperm had significantly reduced 5-methylcytosine at CpG locations of Rfwd2 ( a ), Sfi1 ( b ), Kdm3b ( c ) , Gm52 ( d ) and Rbks ( e ). Means±s.e.m. of five determinations are shown. * P
    Figure Legend Snippet: Folate deficiency alters sperm DNA methylation at genes implicated in development and metabolic processes. Sequenom MassARRAY methylation analysis was performed on selected targets of interest identified by MeDIP-chip as altered in FD versus FS sperm. FD sperm had significantly reduced 5-methylcytosine at CpG locations of Rfwd2 ( a ), Sfi1 ( b ), Kdm3b ( c ) , Gm52 ( d ) and Rbks ( e ). Means±s.e.m. of five determinations are shown. * P

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

    6) Product Images from "Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii"

    Article Title: Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky373

    TgAP2X-5 depletion prevents TgAP2XI-5 binding to a number of promoters. ( A ) ChIP-on-chip data are represented as the log 2 ratio of the hybridization signal given by DNA immunoprecipitated over the signal given by the non-enriched input DNA. The log 2 ratio of each oligonucleotide present on the tiled microarray has been plotted at the respective genomic position. Genes above the horizontal axis are encoded the forward strand whereas those below are encoded the reverse strand. The scale of the Y -axis was kept identical for all representation to allow the comparison of signals. TgAP2XI-5 ChIP-chip assay is represented for the TgAP2XI-5 protein in the parental strain (RH ΔhxgprtΔku80 , green, negative control) or TgAP2XI-5 tagged strain (red, positive control) or TgAP2XI-5 tagged in the iKD TgAP2X-5 strain in presence of ATc (orange). The associated peaks identified by the mPeak algorithm are also provided. A portion of the Toxoplasma gondii chromosome III is represented. The boxed promoter presents a lower enrichment of TgAP2XI-5 in the iKD TgAP2X-5 strain in presence of ATc. ( B ) Representation of the ChIP-chip experiments for a portion of chromosome IV which encompasses the TgROP15 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( C ) Representation of the ChIP-chip experiments for a portion of chromosome III which encompasses the TgROP24 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( D ) Quantitative PCR was performed on the TgAP2XI-5 ChIP assays performed in the iKD TgAP2X-5/TgAP2XI-5 strain (orange), iKD TgAP2X-5 complemented/TgAP2XI-5 strain (yellow), TgAP2XI-5-tagged strain (red) and parental strain (negative control, green). Amplification was carried out on regions within the promoter of each of the six genes listed. The enrichment for corresponding ChIP DNA samples is presented as a percentage of INPUT for each target.
    Figure Legend Snippet: TgAP2X-5 depletion prevents TgAP2XI-5 binding to a number of promoters. ( A ) ChIP-on-chip data are represented as the log 2 ratio of the hybridization signal given by DNA immunoprecipitated over the signal given by the non-enriched input DNA. The log 2 ratio of each oligonucleotide present on the tiled microarray has been plotted at the respective genomic position. Genes above the horizontal axis are encoded the forward strand whereas those below are encoded the reverse strand. The scale of the Y -axis was kept identical for all representation to allow the comparison of signals. TgAP2XI-5 ChIP-chip assay is represented for the TgAP2XI-5 protein in the parental strain (RH ΔhxgprtΔku80 , green, negative control) or TgAP2XI-5 tagged strain (red, positive control) or TgAP2XI-5 tagged in the iKD TgAP2X-5 strain in presence of ATc (orange). The associated peaks identified by the mPeak algorithm are also provided. A portion of the Toxoplasma gondii chromosome III is represented. The boxed promoter presents a lower enrichment of TgAP2XI-5 in the iKD TgAP2X-5 strain in presence of ATc. ( B ) Representation of the ChIP-chip experiments for a portion of chromosome IV which encompasses the TgROP15 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( C ) Representation of the ChIP-chip experiments for a portion of chromosome III which encompasses the TgROP24 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( D ) Quantitative PCR was performed on the TgAP2XI-5 ChIP assays performed in the iKD TgAP2X-5/TgAP2XI-5 strain (orange), iKD TgAP2X-5 complemented/TgAP2XI-5 strain (yellow), TgAP2XI-5-tagged strain (red) and parental strain (negative control, green). Amplification was carried out on regions within the promoter of each of the six genes listed. The enrichment for corresponding ChIP DNA samples is presented as a percentage of INPUT for each target.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Hybridization, Immunoprecipitation, Microarray, Negative Control, Positive Control, Real-time Polymerase Chain Reaction, Amplification

    7) Product Images from "Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells"

    Article Title: Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells

    Journal: Oncotarget

    doi:

    MeDIP-chip analysis of the promoter DNA-methylome of human HEPs, HSCs and LSECs A. Two-dimensional scatter plots of MaxTen values of methylation intensities for all promoters in HEPs, HSCs and LSECs. Genes with a promoter significantly methylated in one cell type are colored; non-significantly methylated genes are shown in gray. B. Browser view of promoter methylation on all chromosomes; right , zoom-in of GFRA3 methylation in HEPs, HSCs and LSECs (log (MeDIP/Input) ratios). Red and blue colors point to methylation peaks and depletions, respectively. C. Venn diagram analysis of numbers of genes with a methylated promoter in HSCs, LSECs and HEPs. D. Most significant GO terms for the methylation ‘core’ and for cell type-specific methylated genes. E. Proportion of genes that are uniquely or commonly methylated between two or more cell types. F. Promoter methylation in HSCs, LSECs and HEPs relative to CD34 + bone marrow progenitors. Percentage of methylated genes in cell types shown on the x-axis that are also methylated in cell types shown on the y-axis.
    Figure Legend Snippet: MeDIP-chip analysis of the promoter DNA-methylome of human HEPs, HSCs and LSECs A. Two-dimensional scatter plots of MaxTen values of methylation intensities for all promoters in HEPs, HSCs and LSECs. Genes with a promoter significantly methylated in one cell type are colored; non-significantly methylated genes are shown in gray. B. Browser view of promoter methylation on all chromosomes; right , zoom-in of GFRA3 methylation in HEPs, HSCs and LSECs (log (MeDIP/Input) ratios). Red and blue colors point to methylation peaks and depletions, respectively. C. Venn diagram analysis of numbers of genes with a methylated promoter in HSCs, LSECs and HEPs. D. Most significant GO terms for the methylation ‘core’ and for cell type-specific methylated genes. E. Proportion of genes that are uniquely or commonly methylated between two or more cell types. F. Promoter methylation in HSCs, LSECs and HEPs relative to CD34 + bone marrow progenitors. Percentage of methylated genes in cell types shown on the x-axis that are also methylated in cell types shown on the y-axis.

    Techniques Used: Methylated DNA Immunoprecipitation, Chromatin Immunoprecipitation, Methylation

    Culture-induced HSC activation reprograms promoter DNA methylation A. Venn diagram analysis of the number of genes with a methylated promoter in qHSCs and aHSCs. B. Browser views of promoter methylation profiles (log (MeDIP/Input) ratios) for indicated genes in qHSCs and aHSCs. Red and blue colors point to methylation peaks and depletions, respectively. C. Heatmap of genes up-regulated and hypo-methylated after HSC activation. D. Boxwhisker plot of ACTG2 expression in qHSCs and aHSCs. E. Bisulfite sequencing analysis of CpG methylation in the ACTG2 promoter in qHSCs and aHSCs. Four CpGs are examined (columns) in 5 sequenced clones (rows). ● methylated CpG; ○ unmethylated CpG. F. Heatmap of genes down-regulated and hyper-methylated after HSC activation. G. Boxwhisker plot of APOB expression in qHSCs and aHSCs. H. Bisulfite sequencing analysis of CpG methylation in the APOB promoter in qHSCs and aHSCs. Five CpGs were analyzed.
    Figure Legend Snippet: Culture-induced HSC activation reprograms promoter DNA methylation A. Venn diagram analysis of the number of genes with a methylated promoter in qHSCs and aHSCs. B. Browser views of promoter methylation profiles (log (MeDIP/Input) ratios) for indicated genes in qHSCs and aHSCs. Red and blue colors point to methylation peaks and depletions, respectively. C. Heatmap of genes up-regulated and hypo-methylated after HSC activation. D. Boxwhisker plot of ACTG2 expression in qHSCs and aHSCs. E. Bisulfite sequencing analysis of CpG methylation in the ACTG2 promoter in qHSCs and aHSCs. Four CpGs are examined (columns) in 5 sequenced clones (rows). ● methylated CpG; ○ unmethylated CpG. F. Heatmap of genes down-regulated and hyper-methylated after HSC activation. G. Boxwhisker plot of APOB expression in qHSCs and aHSCs. H. Bisulfite sequencing analysis of CpG methylation in the APOB promoter in qHSCs and aHSCs. Five CpGs were analyzed.

    Techniques Used: Activation Assay, DNA Methylation Assay, Methylation, Methylated DNA Immunoprecipitation, Expressing, Methylation Sequencing, CpG Methylation Assay

    8) Product Images from "Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus"

    Article Title: Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000909

    Establishment of Chromatin immunoprecipitation. (A) Nuclear localization of MoCRZ1 was visualized in the eGFP tagged strain under the native promoter. FK506 blocks nuclear localization of MoCRZ1::eGFP. Bar indicates 10 µm. (B) ChIP-chip experimental design to identify MoCRZ1 targets activated by calcium treatment. The Ca 2+ /FK506 treated sample served as the negative control treatment. (C) RT-PCR to verify that PMC1 was up-regulated in the Ca 2+ treated but not in Ca 2+ /FK506 treated sample. (D) Quantitative PCR was conducted with DNA after ChIP with antiGFP antibody. 30% input DNA collected prior to pull down was used as control. 1 µl each of ChIPed and input DNA was used for real-time PCR. Fold changes were calculated by 2 ΔΔCt , where ΔΔCt = (Ct input DNA -Ct ChIPed DNA ) Ca2+ treated sample - (Ct input DNA -Ct ChIPed DNA ) Ca2+/FK506 treated sample .
    Figure Legend Snippet: Establishment of Chromatin immunoprecipitation. (A) Nuclear localization of MoCRZ1 was visualized in the eGFP tagged strain under the native promoter. FK506 blocks nuclear localization of MoCRZ1::eGFP. Bar indicates 10 µm. (B) ChIP-chip experimental design to identify MoCRZ1 targets activated by calcium treatment. The Ca 2+ /FK506 treated sample served as the negative control treatment. (C) RT-PCR to verify that PMC1 was up-regulated in the Ca 2+ treated but not in Ca 2+ /FK506 treated sample. (D) Quantitative PCR was conducted with DNA after ChIP with antiGFP antibody. 30% input DNA collected prior to pull down was used as control. 1 µl each of ChIPed and input DNA was used for real-time PCR. Fold changes were calculated by 2 ΔΔCt , where ΔΔCt = (Ct input DNA -Ct ChIPed DNA ) Ca2+ treated sample - (Ct input DNA -Ct ChIPed DNA ) Ca2+/FK506 treated sample .

    Techniques Used: Chromatin Immunoprecipitation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    9) Product Images from "Elevation of Il6 is associated with disturbed let-7 biogenesis in a genetic model of depression"

    Article Title: Elevation of Il6 is associated with disturbed let-7 biogenesis in a genetic model of depression

    Journal: Translational Psychiatry

    doi: 10.1038/tp.2016.136

    Gene and miRNA expression levels were measured in the prefrontal cortex of FSL with/without access to running wheel (FSL-runners versus FSL-controls) using RT-PCR. ( a ) Physical activity normalized Il6 levels in the FSL-runner group but had no effect on Lin28b and Drosha mRNA levels. ( b ) In line with an Il6 reduction in the FSL rats that were running, let-7i and miR-98 showed significantly increased expression. Specifically, upregulation of let-7i was associated with primary let-7i overexpression ( c ). ( d ) Chromatin immunoprecipitation (ChIP) showed an increased histone H4 acetylation (H4ac) but not histone H3 acetylation (H3ac) within the pri-let-7i promoter of the FSL-runners. Gene expression data were presented as relative quantifications (R.Q.), two reference genes ( Gapdh and Ppia ) were used for normalization of Il6 , Lin28b , Drosha and the primary miRNA genes. Rnu5g was used for normalization of mature miRNAs. ChIP data are presented as percentage of genomic input DNA. Data are presented as group means±s.e.m. For all figures: n =5–7 animals per group, * P
    Figure Legend Snippet: Gene and miRNA expression levels were measured in the prefrontal cortex of FSL with/without access to running wheel (FSL-runners versus FSL-controls) using RT-PCR. ( a ) Physical activity normalized Il6 levels in the FSL-runner group but had no effect on Lin28b and Drosha mRNA levels. ( b ) In line with an Il6 reduction in the FSL rats that were running, let-7i and miR-98 showed significantly increased expression. Specifically, upregulation of let-7i was associated with primary let-7i overexpression ( c ). ( d ) Chromatin immunoprecipitation (ChIP) showed an increased histone H4 acetylation (H4ac) but not histone H3 acetylation (H3ac) within the pri-let-7i promoter of the FSL-runners. Gene expression data were presented as relative quantifications (R.Q.), two reference genes ( Gapdh and Ppia ) were used for normalization of Il6 , Lin28b , Drosha and the primary miRNA genes. Rnu5g was used for normalization of mature miRNAs. ChIP data are presented as percentage of genomic input DNA. Data are presented as group means±s.e.m. For all figures: n =5–7 animals per group, * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Over Expression, Chromatin Immunoprecipitation

    10) Product Images from "Effects of Sulforaphane and 3,3?-Diindolylmethane on Genome-Wide Promoter Methylation in Normal Prostate Epithelial Cells and Prostate Cancer Cells"

    Article Title: Effects of Sulforaphane and 3,3?-Diindolylmethane on Genome-Wide Promoter Methylation in Normal Prostate Epithelial Cells and Prostate Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086787

    Genome-wide promoter methylation profiling comparing normal prostate epithelial cells and prostate cancer cells. (A) Hierarchical clustering analysis of probes with significant scaled log 2 ratio in PrEC, LnCAP, and PC3 cells. Green and red bars represent individual probes with significant decreased and increased methylation, respectively, of MeDIP DNA samples relative to input DNA samples. Data represent the top 1,000 most significant probes within each cell line. (B) A comparison of methylation changes in LnCAP and PC3 cells relative to PrEC cells. Significant methylated probes in LnCAP and PC3 cells were compared to PrEC, and the distribution of probes with significant log2 fold-changes are shown. (C) Average methylation level in individual genes in LnCAP and PC3 cells compared to PrEC. Log2 fold-change per gene was determined by averaging the log2 fold-change of all differentially methylated probes assigned to each gene, and represented as box and whisker plots. Number above the bar denotes the number of genes in each group. Whiskers represent maximum and minimum values, and “+” represents mean value.
    Figure Legend Snippet: Genome-wide promoter methylation profiling comparing normal prostate epithelial cells and prostate cancer cells. (A) Hierarchical clustering analysis of probes with significant scaled log 2 ratio in PrEC, LnCAP, and PC3 cells. Green and red bars represent individual probes with significant decreased and increased methylation, respectively, of MeDIP DNA samples relative to input DNA samples. Data represent the top 1,000 most significant probes within each cell line. (B) A comparison of methylation changes in LnCAP and PC3 cells relative to PrEC cells. Significant methylated probes in LnCAP and PC3 cells were compared to PrEC, and the distribution of probes with significant log2 fold-changes are shown. (C) Average methylation level in individual genes in LnCAP and PC3 cells compared to PrEC. Log2 fold-change per gene was determined by averaging the log2 fold-change of all differentially methylated probes assigned to each gene, and represented as box and whisker plots. Number above the bar denotes the number of genes in each group. Whiskers represent maximum and minimum values, and “+” represents mean value.

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

    11) Product Images from "Induction of altered epigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricted diet during pregnancy suggests that reduced DNA methyltransferase-1 expression is involved in impaired DNA methylation and changes in histone modifications"

    Article Title: Induction of altered epigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricted diet during pregnancy suggests that reduced DNA methyltransferase-1 expression is involved in impaired DNA methylation and changes in histone modifications

    Journal:

    doi: 10.1017/S000711450769196X

    mRNA expression of DNA methyltransferases (Dnmt (s)) in liver from 34 day-old offspring of rats fed either a control or protein-restricted (PR), or the PR diet supplemented with folic acid (PRF) diet during pregnancy. Data from RT PCR analysis are mean
    Figure Legend Snippet: mRNA expression of DNA methyltransferases (Dnmt (s)) in liver from 34 day-old offspring of rats fed either a control or protein-restricted (PR), or the PR diet supplemented with folic acid (PRF) diet during pregnancy. Data from RT PCR analysis are mean

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice"

    Article Title: Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018216

    Overview of study design and data integration. ( A ) Experimental strategy for the identification and integration of PB-induced expression and epigenetic perturbations in target (liver) and non-target (kidney) tissues. RNA, DNA and chromatin was extracted from liver and kidney samples of control and PB-treated (4-week, 0.05% in drinking water) B6C3F1 male mice (n = 10 per group) and analyzed through the different profiling methodologies and platforms indicated. Abbreviations: liquid chromatography-mass spectrometry (LC-MS), Methylated DNA immunoprecipitation (MeDIP), Chromatin immunoprecipitation (ChIP), quantitative real-time PCR (qPCR). ( B ) Summary of bioinformatic data integration strategy. For each annotated gene present on gene expression and promoter arrays, the expression and DNA methylation values were mapped to the genome and correlated to examine the functional links between expression and methylation levels at individual loci upon phenobarbital treatment. For loci of interest the abundance of selected chromatin marks were quantified. Coverage of the promoter array (1.8 kb per promoter: 500 bp downstream and 1300 bp upstream of the transcriptional start site (TSS)) is shown. For the methylation analysis a window of 100 bp downstream and 800 bp upstream of the TSS was used. The figure shows an exemplary gene that upon treatment loses DNA methylation and gets expressed.
    Figure Legend Snippet: Overview of study design and data integration. ( A ) Experimental strategy for the identification and integration of PB-induced expression and epigenetic perturbations in target (liver) and non-target (kidney) tissues. RNA, DNA and chromatin was extracted from liver and kidney samples of control and PB-treated (4-week, 0.05% in drinking water) B6C3F1 male mice (n = 10 per group) and analyzed through the different profiling methodologies and platforms indicated. Abbreviations: liquid chromatography-mass spectrometry (LC-MS), Methylated DNA immunoprecipitation (MeDIP), Chromatin immunoprecipitation (ChIP), quantitative real-time PCR (qPCR). ( B ) Summary of bioinformatic data integration strategy. For each annotated gene present on gene expression and promoter arrays, the expression and DNA methylation values were mapped to the genome and correlated to examine the functional links between expression and methylation levels at individual loci upon phenobarbital treatment. For loci of interest the abundance of selected chromatin marks were quantified. Coverage of the promoter array (1.8 kb per promoter: 500 bp downstream and 1300 bp upstream of the transcriptional start site (TSS)) is shown. For the methylation analysis a window of 100 bp downstream and 800 bp upstream of the TSS was used. The figure shows an exemplary gene that upon treatment loses DNA methylation and gets expressed.

    Techniques Used: Expressing, Mouse Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Functional Assay

    MeDIP-promoter array profiling of the methylome in the liver of control and PB treated B6C3F1 mice. ( A ) An antibody directed against 5-methyl-cytosine (5mC) was used for immunoprecipitation of methylated DNA. Control sequences that are highly methylated (IAP, H19 ICR) or lack CpGs (CSa) were selected as controls for the MeDIP experiment prior to and following whole genome amplification (WGA). The relative enrichment in the bound over input fractions for 10 individual biological replicates was measured by qPCR. ( B ) Methylation comparison between liver of control and PB-treated mice (average log 2 (IP/total) for replicates) in all 23,428 Nimblegen probe sets. The colors indicate the CpG island class for those probe sets where the log 2 methylation ratio of PB-treated vs. control (difference in M-value) is significant (p≤0.01 and an absolute log 2 fold change of ≥0.2, 28 probe sets), non-differentially methylated regions are indicated in grey. A circle highlights Cyp2b10 .
    Figure Legend Snippet: MeDIP-promoter array profiling of the methylome in the liver of control and PB treated B6C3F1 mice. ( A ) An antibody directed against 5-methyl-cytosine (5mC) was used for immunoprecipitation of methylated DNA. Control sequences that are highly methylated (IAP, H19 ICR) or lack CpGs (CSa) were selected as controls for the MeDIP experiment prior to and following whole genome amplification (WGA). The relative enrichment in the bound over input fractions for 10 individual biological replicates was measured by qPCR. ( B ) Methylation comparison between liver of control and PB-treated mice (average log 2 (IP/total) for replicates) in all 23,428 Nimblegen probe sets. The colors indicate the CpG island class for those probe sets where the log 2 methylation ratio of PB-treated vs. control (difference in M-value) is significant (p≤0.01 and an absolute log 2 fold change of ≥0.2, 28 probe sets), non-differentially methylated regions are indicated in grey. A circle highlights Cyp2b10 .

    Techniques Used: Methylated DNA Immunoprecipitation, Mouse Assay, Immunoprecipitation, Methylation, Whole Genome Amplification, Real-time Polymerase Chain Reaction

    Cyp2b10 is selectively DNA demethylated and over expressed in the liver of PB-treated B6C3F1 mice. ( A ) RT-qPCR analysis of Cy2b10 expression in the liver and the kidney of control and PB-treated animals. Fold changes are indicated relative to 18S RNA expression levels. ( B ) MeDIP-qPCR validation of Nimblegen data. Positive (H19 ICR, IAP), negative (CSa, Intergenic3, Hprt, Gapdh) and selected regions identified on the promoter array were assessed by qPCR from WGA amplified MeDIP and input DNA samples prepared from the liver of 6 control and PB-treated mice. qPCR identified selective demethylation at Cyp2b10 TSS, both in the promoter and first intron (location of PCR amplicons is shown in Figure 3C). Relative enrichment (IP/Input) for DNA methylation of 6 individual biological replicates is shown. ( C ) Bisulfite sequencing at Cyp2b10 first intron. Sequenced area is shown by the two arrows in the schematic gene map. Each line represents the sequence of a single clone. CpGs are shown as white (unmethylated) or black (methylated) circles. The values above summarizes the overall methylation level of this region (percentage of methylated CpG in all sequenced clones) ( D ) Quantitative DNA methylation analysis by pyrosequencing of two Cyp2b10 promoter CpG sites (CpG1: -914 and CpG2: -886 indicated in (C)) in the liver and kidney of control and PB-treated animals. Standard deviation was calculated from 10 biological replicates. Primers/genomic regions used for bisulfite sequencing are available in Figure S9.
    Figure Legend Snippet: Cyp2b10 is selectively DNA demethylated and over expressed in the liver of PB-treated B6C3F1 mice. ( A ) RT-qPCR analysis of Cy2b10 expression in the liver and the kidney of control and PB-treated animals. Fold changes are indicated relative to 18S RNA expression levels. ( B ) MeDIP-qPCR validation of Nimblegen data. Positive (H19 ICR, IAP), negative (CSa, Intergenic3, Hprt, Gapdh) and selected regions identified on the promoter array were assessed by qPCR from WGA amplified MeDIP and input DNA samples prepared from the liver of 6 control and PB-treated mice. qPCR identified selective demethylation at Cyp2b10 TSS, both in the promoter and first intron (location of PCR amplicons is shown in Figure 3C). Relative enrichment (IP/Input) for DNA methylation of 6 individual biological replicates is shown. ( C ) Bisulfite sequencing at Cyp2b10 first intron. Sequenced area is shown by the two arrows in the schematic gene map. Each line represents the sequence of a single clone. CpGs are shown as white (unmethylated) or black (methylated) circles. The values above summarizes the overall methylation level of this region (percentage of methylated CpG in all sequenced clones) ( D ) Quantitative DNA methylation analysis by pyrosequencing of two Cyp2b10 promoter CpG sites (CpG1: -914 and CpG2: -886 indicated in (C)) in the liver and kidney of control and PB-treated animals. Standard deviation was calculated from 10 biological replicates. Primers/genomic regions used for bisulfite sequencing are available in Figure S9.

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Expressing, RNA Expression, Methylated DNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Whole Genome Amplification, Amplification, Polymerase Chain Reaction, DNA Methylation Assay, Methylation Sequencing, Sequencing, Methylation, Standard Deviation

    13) Product Images from "Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders"

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131144

    The levels of H3K27me3 at promoter regions in GMPs. Visualization of ChIP-seq data of the H3K27me3 levels of several developmental regulator genes ( Hoxd locus, Gata4 , and Pax5 ), tumor suppressor genes ( Cdkn2a and Cdkn2b ) and oncogenes ( Hmga2 and Pbx3 ) in GMPs from WT, Tet2 KD/KD , Ezh2 Δ/Δ , and Tet2 KD/KD Ezh2 Δ/Δ mice at 4 mo after transplantation using the Integrative Genomics Viewer (IGV). Schematic diagram of these gene loci indicates their genomic structures. Exons and untranslated regions are demarcated by large and small black boxes, respectively. Data of ChIP analyses at the promoters of selected genes are also depicted. Quantitative ChIP analyses of GMPs from WT and Ezh2 Δ/Δ mice at 6 mo after transplantation were performed. The relative amounts of immunoprecipitated DNA are depicted as a percentage of input DNA. N.D. indicates not detected. The data are shown as the mean ± SEM for triplicate analyses. Regions amplified from the precipitated DNA by site-specific quantitative PCR are indicated by arrows. *, P
    Figure Legend Snippet: The levels of H3K27me3 at promoter regions in GMPs. Visualization of ChIP-seq data of the H3K27me3 levels of several developmental regulator genes ( Hoxd locus, Gata4 , and Pax5 ), tumor suppressor genes ( Cdkn2a and Cdkn2b ) and oncogenes ( Hmga2 and Pbx3 ) in GMPs from WT, Tet2 KD/KD , Ezh2 Δ/Δ , and Tet2 KD/KD Ezh2 Δ/Δ mice at 4 mo after transplantation using the Integrative Genomics Viewer (IGV). Schematic diagram of these gene loci indicates their genomic structures. Exons and untranslated regions are demarcated by large and small black boxes, respectively. Data of ChIP analyses at the promoters of selected genes are also depicted. Quantitative ChIP analyses of GMPs from WT and Ezh2 Δ/Δ mice at 6 mo after transplantation were performed. The relative amounts of immunoprecipitated DNA are depicted as a percentage of input DNA. N.D. indicates not detected. The data are shown as the mean ± SEM for triplicate analyses. Regions amplified from the precipitated DNA by site-specific quantitative PCR are indicated by arrows. *, P

    Techniques Used: Chromatin Immunoprecipitation, Mouse Assay, Transplantation Assay, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    14) Product Images from "Evaluation of affinity-based genome-wide DNA methylation data: Effects of CpG density, amplification bias, and copy number variation"

    Article Title: Evaluation of affinity-based genome-wide DNA methylation data: Effects of CpG density, amplification bias, and copy number variation

    Journal: Genome Research

    doi: 10.1101/gr.110601.110

    ( A ) Schematic showing the capture of methylated DNA into populations of single-stranded (MeDIP) or double-stranded (MBDCap) fragments. ( B ) Summarized probe intensities for enrichment of fully methylated DNA with MeDIP and two variations of MethylMiner-based
    Figure Legend Snippet: ( A ) Schematic showing the capture of methylated DNA into populations of single-stranded (MeDIP) or double-stranded (MBDCap) fragments. ( B ) Summarized probe intensities for enrichment of fully methylated DNA with MeDIP and two variations of MethylMiner-based

    Techniques Used: Methylation, Methylated DNA Immunoprecipitation

    15) Product Images from "Developmental features of DNA methylation during activation of the embryonic zebrafish genome"

    Article Title: Developmental features of DNA methylation during activation of the embryonic zebrafish genome

    Journal: Genome Biology

    doi: 10.1186/gb-2012-13-7-r65

    Promoter DNA methylation states during the transition through the MBT period . (a) MeDIP-chip profiles of DNA methylation in tiled regions spanning a housekeeping gene ( bact1 ) and developmentally regulated genes ( klf4 , pou5f1 , fez1 ) (log2 MeDIP/input ratios), in pre-MBT, MBT, and post-MBT embryos and in the ZF4 fibroblast cell line. Red arrows in the upper track point to regions analyzed by bisulfite sequencing in (b). (b) Two-dimensional scatter plots of MaxSixty values for MeDIP log 2 signal intensities at indicated developmental stages (pairwise) and in ZF4 cells. Average MaxSixty values for both MeDIP replicates are plotted for each stage. Data points are colored to indicate classification according to peak calling algorithm, to show methylated promoters in one only (purple, green) or both (blue) stages. (c) Bisulfite sequencing validation of MeDIP-chip data shown in (a); 5' to 3' orientation; filled circles indicate methylated cytosine; empty circles indicate unmethylated cytosine. (d) Numbers of methylated genes pre-MBT, MBT and post-MBT. Color reflects genes whose methylation is maintained between stages.
    Figure Legend Snippet: Promoter DNA methylation states during the transition through the MBT period . (a) MeDIP-chip profiles of DNA methylation in tiled regions spanning a housekeeping gene ( bact1 ) and developmentally regulated genes ( klf4 , pou5f1 , fez1 ) (log2 MeDIP/input ratios), in pre-MBT, MBT, and post-MBT embryos and in the ZF4 fibroblast cell line. Red arrows in the upper track point to regions analyzed by bisulfite sequencing in (b). (b) Two-dimensional scatter plots of MaxSixty values for MeDIP log 2 signal intensities at indicated developmental stages (pairwise) and in ZF4 cells. Average MaxSixty values for both MeDIP replicates are plotted for each stage. Data points are colored to indicate classification according to peak calling algorithm, to show methylated promoters in one only (purple, green) or both (blue) stages. (c) Bisulfite sequencing validation of MeDIP-chip data shown in (a); 5' to 3' orientation; filled circles indicate methylated cytosine; empty circles indicate unmethylated cytosine. (d) Numbers of methylated genes pre-MBT, MBT and post-MBT. Color reflects genes whose methylation is maintained between stages.

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

    16) Product Images from "Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining"

    Article Title: Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

    Journal: bioRxiv

    doi: 10.1101/754242

    The native A. baylyi ADP1 CRISPR-Cas system is active and can be retargeted. Scheme for reprogramming the A. baylyi CRISPR array. First, a “CRISPR-Ready” strain is created by performing a Golden Transformation that replaces the entire native spacer array with a tdk - kanR cassette. Then, a second Golden Transformation can be used to add a rescue cassette that contains one or more designed spacers under control of the native gene expression signals. Single-spacer replacement cassette design. Synthetic double-stranded DNA encoding the spacer and surrounding repeats is combined with PCR products corresponding to the flanking genome homology upstream (U) and downstream (D) using BsmBI Golden Gate assembly. The inset shows the DNA sequence that is targeted for cleavage with the protospacer adjacent motif (PAM) typical of type-I CRISPR-Cas systems. (C) Reprogrammed CRISPR-Cas system restricts transformation of foreign DNA. Frequencies of transformants of genomic DNA from A. baylyi ADP1 donor that has an integrated spectinomycin resistance gene ( specR ) were used to judge whether targeting a spacer to this sequence in a recipient strain prevented its acquisition. WT is wild type ADP1-ISx and CR is the CRISPR-Ready derivative of this strain. S1 and S2 are controls with spacers that match the specR sequence but in incorrect PAM contexts. S3 is an on-target specR spacer with the correct PAM. Error bars are estimated 95% confidence intervals.
    Figure Legend Snippet: The native A. baylyi ADP1 CRISPR-Cas system is active and can be retargeted. Scheme for reprogramming the A. baylyi CRISPR array. First, a “CRISPR-Ready” strain is created by performing a Golden Transformation that replaces the entire native spacer array with a tdk - kanR cassette. Then, a second Golden Transformation can be used to add a rescue cassette that contains one or more designed spacers under control of the native gene expression signals. Single-spacer replacement cassette design. Synthetic double-stranded DNA encoding the spacer and surrounding repeats is combined with PCR products corresponding to the flanking genome homology upstream (U) and downstream (D) using BsmBI Golden Gate assembly. The inset shows the DNA sequence that is targeted for cleavage with the protospacer adjacent motif (PAM) typical of type-I CRISPR-Cas systems. (C) Reprogrammed CRISPR-Cas system restricts transformation of foreign DNA. Frequencies of transformants of genomic DNA from A. baylyi ADP1 donor that has an integrated spectinomycin resistance gene ( specR ) were used to judge whether targeting a spacer to this sequence in a recipient strain prevented its acquisition. WT is wild type ADP1-ISx and CR is the CRISPR-Ready derivative of this strain. S1 and S2 are controls with spacers that match the specR sequence but in incorrect PAM contexts. S3 is an on-target specR spacer with the correct PAM. Error bars are estimated 95% confidence intervals.

    Techniques Used: CRISPR, Transformation Assay, Expressing, Polymerase Chain Reaction, Sequencing

    Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.
    Figure Legend Snippet: Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, Selection, Plasmid Preparation

    Self-targeting spacers can be used to assure deletions and create a CRISPR-Lock. CRISPR-Ready variants of wild-type ADP1-ISx (WT) and two multiple gene deletions strains (MGD2 and MGD18) were transformed with different spacers to assess the presence of a sequence located within the putatively deleted region. N1 added back the first spacer sequence from the native CRISPR array. It serves as a control because it does not target any sequence in the A. baylyi genome. T2 and T18 are spacers that match sequences in the ADP1-ISx genome that are within the regions deleted in the corresponding MGD strains. For each strain-spacer combination tested, 10 AZT R colonies were isolated after transformation with the single-spacer replacement DNA. Successful integration of the spacer can only occur if the targeted region is not present in the recipient strain’s genome. It results in these AZT R colonies also becoming Kan S . If integration of the spacer is lethal, then AZT R colonies are expected to have mutations that inactivate the tdk gene and remain Kan R , as illustrated in the upper panel. Strains with successful spacer integrations from the MGD2+T2 and MGD18+T18 transformations have a “CRISPR-Lock” in their genomes that can prevent re-acquisition of the deleted regions when combining multiple deletions in further stages of the genome streamlining project.
    Figure Legend Snippet: Self-targeting spacers can be used to assure deletions and create a CRISPR-Lock. CRISPR-Ready variants of wild-type ADP1-ISx (WT) and two multiple gene deletions strains (MGD2 and MGD18) were transformed with different spacers to assess the presence of a sequence located within the putatively deleted region. N1 added back the first spacer sequence from the native CRISPR array. It serves as a control because it does not target any sequence in the A. baylyi genome. T2 and T18 are spacers that match sequences in the ADP1-ISx genome that are within the regions deleted in the corresponding MGD strains. For each strain-spacer combination tested, 10 AZT R colonies were isolated after transformation with the single-spacer replacement DNA. Successful integration of the spacer can only occur if the targeted region is not present in the recipient strain’s genome. It results in these AZT R colonies also becoming Kan S . If integration of the spacer is lethal, then AZT R colonies are expected to have mutations that inactivate the tdk gene and remain Kan R , as illustrated in the upper panel. Strains with successful spacer integrations from the MGD2+T2 and MGD18+T18 transformations have a “CRISPR-Lock” in their genomes that can prevent re-acquisition of the deleted regions when combining multiple deletions in further stages of the genome streamlining project.

    Techniques Used: CRISPR, Transformation Assay, Sequencing, Isolation

    17) Product Images from "Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii"

    Article Title: Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky373

    TgAP2X-5 depletion prevents TgAP2XI-5 binding to a number of promoters. ( A ) ChIP-on-chip data are represented as the log 2 ratio of the hybridization signal given by DNA immunoprecipitated over the signal given by the non-enriched input DNA. The log 2 ratio of each oligonucleotide present on the tiled microarray has been plotted at the respective genomic position. Genes above the horizontal axis are encoded the forward strand whereas those below are encoded the reverse strand. The scale of the Y -axis was kept identical for all representation to allow the comparison of signals. TgAP2XI-5 ChIP-chip assay is represented for the TgAP2XI-5 protein in the parental strain (RH ΔhxgprtΔku80 , green, negative control) or TgAP2XI-5 tagged strain (red, positive control) or TgAP2XI-5 tagged in the iKD TgAP2X-5 strain in presence of ATc (orange). The associated peaks identified by the mPeak algorithm are also provided. A portion of the Toxoplasma gondii chromosome III is represented. The boxed promoter presents a lower enrichment of TgAP2XI-5 in the iKD TgAP2X-5 strain in presence of ATc. ( B ) Representation of the ChIP-chip experiments for a portion of chromosome IV which encompasses the TgROP15 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( C ) Representation of the ChIP-chip experiments for a portion of chromosome III which encompasses the TgROP24 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( D ) Quantitative PCR was performed on the TgAP2XI-5 ChIP assays performed in the iKD TgAP2X-5/TgAP2XI-5 strain (orange), iKD TgAP2X-5 complemented/TgAP2XI-5 strain (yellow), TgAP2XI-5-tagged strain (red) and parental strain (negative control, green). Amplification was carried out on regions within the promoter of each of the six genes listed. The enrichment for corresponding ChIP DNA samples is presented as a percentage of INPUT for each target.
    Figure Legend Snippet: TgAP2X-5 depletion prevents TgAP2XI-5 binding to a number of promoters. ( A ) ChIP-on-chip data are represented as the log 2 ratio of the hybridization signal given by DNA immunoprecipitated over the signal given by the non-enriched input DNA. The log 2 ratio of each oligonucleotide present on the tiled microarray has been plotted at the respective genomic position. Genes above the horizontal axis are encoded the forward strand whereas those below are encoded the reverse strand. The scale of the Y -axis was kept identical for all representation to allow the comparison of signals. TgAP2XI-5 ChIP-chip assay is represented for the TgAP2XI-5 protein in the parental strain (RH ΔhxgprtΔku80 , green, negative control) or TgAP2XI-5 tagged strain (red, positive control) or TgAP2XI-5 tagged in the iKD TgAP2X-5 strain in presence of ATc (orange). The associated peaks identified by the mPeak algorithm are also provided. A portion of the Toxoplasma gondii chromosome III is represented. The boxed promoter presents a lower enrichment of TgAP2XI-5 in the iKD TgAP2X-5 strain in presence of ATc. ( B ) Representation of the ChIP-chip experiments for a portion of chromosome IV which encompasses the TgROP15 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( C ) Representation of the ChIP-chip experiments for a portion of chromosome III which encompasses the TgROP24 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( D ) Quantitative PCR was performed on the TgAP2XI-5 ChIP assays performed in the iKD TgAP2X-5/TgAP2XI-5 strain (orange), iKD TgAP2X-5 complemented/TgAP2XI-5 strain (yellow), TgAP2XI-5-tagged strain (red) and parental strain (negative control, green). Amplification was carried out on regions within the promoter of each of the six genes listed. The enrichment for corresponding ChIP DNA samples is presented as a percentage of INPUT for each target.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Hybridization, Immunoprecipitation, Microarray, Negative Control, Positive Control, Real-time Polymerase Chain Reaction, Amplification

    18) Product Images from "Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin"

    Article Title: Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007377

    Physical association of the putative helicase (K2ORF4p), mRNA capping enzyme (K2ORF3p), and the large RNAP subunit (K2ORF6p) of the yeast VLEs with VLE-specific DNA. (A) Western blot of HA-K2ORF4p that was affinity-purified from lysates of IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. The strains used are indicated above the lanes. The antibody used is indicated on the left hand side of the strip. The protein detected is indicated on the right hand side of the strip. (B) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K1ORF3 , K2ORF3 ) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. Samples of individually performed gene-specific PCRs were analysed in 2.5% agarose gel stained with ethidium bromide. The identity of the bands (genes) is indicated on the right. M, DNA molecular mass marker (GeneRuler 100 bp Plus DNA Ladder, Fermentas). The respective values are indicated on the left. (C) Western blot of K2ORF3p-HA that was affinity-purified from lysates of IFO1267_pRKL2-14 (K2ORF3p-HA) and IFO1267 (control) cells. (D) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K2ORF3 , K2ORF6 ) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-14 and IFO1267 cells. (E) Western blot of yEGFP3-K2ORF6p that was affinity-purified from lysates of IFO1267_pRKL2-4 (yEGFP3-K2ORF6p) and IFO1267 (control) cells. (F) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K2ORF3 , K2ORF6 ) DNA in chromatin immunoprecipitated using GFP-Trap agarose beads from IFO1267_pRKL2-4 and IFO1267 cells.
    Figure Legend Snippet: Physical association of the putative helicase (K2ORF4p), mRNA capping enzyme (K2ORF3p), and the large RNAP subunit (K2ORF6p) of the yeast VLEs with VLE-specific DNA. (A) Western blot of HA-K2ORF4p that was affinity-purified from lysates of IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. The strains used are indicated above the lanes. The antibody used is indicated on the left hand side of the strip. The protein detected is indicated on the right hand side of the strip. (B) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K1ORF3 , K2ORF3 ) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. Samples of individually performed gene-specific PCRs were analysed in 2.5% agarose gel stained with ethidium bromide. The identity of the bands (genes) is indicated on the right. M, DNA molecular mass marker (GeneRuler 100 bp Plus DNA Ladder, Fermentas). The respective values are indicated on the left. (C) Western blot of K2ORF3p-HA that was affinity-purified from lysates of IFO1267_pRKL2-14 (K2ORF3p-HA) and IFO1267 (control) cells. (D) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K2ORF3 , K2ORF6 ) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-14 and IFO1267 cells. (E) Western blot of yEGFP3-K2ORF6p that was affinity-purified from lysates of IFO1267_pRKL2-4 (yEGFP3-K2ORF6p) and IFO1267 (control) cells. (F) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K2ORF3 , K2ORF6 ) DNA in chromatin immunoprecipitated using GFP-Trap agarose beads from IFO1267_pRKL2-4 and IFO1267 cells.

    Techniques Used: Western Blot, Affinity Purification, Stripping Membranes, Polymerase Chain Reaction, Activated Clotting Time Assay, Immunoprecipitation, Agarose Gel Electrophoresis, Staining, Marker

    19) Product Images from "Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients"

    Article Title: Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddz285

    Targeted NOTCH3 exon exclusion using ASOs and CRISPR/Cas9 in vitro . ( A ) RT-PCR analysis of VSMCs after transfection with an ASO targeting exon 9, showing a band at ~ 300 bp, corresponding to the expected fragment size after exon 9 skipping, which was confirmed to be a correct exon 9 skip with Sanger sequencing. ( B ) Guide RNAs targeting NOTCH3 introns 8 and 9 were used to delete exon 9 from the genomic DNA of HEK293 cells. RT-PCR analysis confirmed exon 9 exclusion at the RNA level. ( C ) Guide RNAs targeting intron 3 and intron 5 were used to delete exon 4 and exon 5 from the genomic DNA from control (left lane) and CADASIL patient-derived VSMCs (right lane), which was confirmed with Sanger sequencing. FL, full length; −RT, condition without reverse transcriptase; Δex9, PCR product lacking exon 9; Δex4–5, PCR product lacking exon 4–5; arrowhead, aspecific genomic PCR product.
    Figure Legend Snippet: Targeted NOTCH3 exon exclusion using ASOs and CRISPR/Cas9 in vitro . ( A ) RT-PCR analysis of VSMCs after transfection with an ASO targeting exon 9, showing a band at ~ 300 bp, corresponding to the expected fragment size after exon 9 skipping, which was confirmed to be a correct exon 9 skip with Sanger sequencing. ( B ) Guide RNAs targeting NOTCH3 introns 8 and 9 were used to delete exon 9 from the genomic DNA of HEK293 cells. RT-PCR analysis confirmed exon 9 exclusion at the RNA level. ( C ) Guide RNAs targeting intron 3 and intron 5 were used to delete exon 4 and exon 5 from the genomic DNA from control (left lane) and CADASIL patient-derived VSMCs (right lane), which was confirmed with Sanger sequencing. FL, full length; −RT, condition without reverse transcriptase; Δex9, PCR product lacking exon 9; Δex4–5, PCR product lacking exon 4–5; arrowhead, aspecific genomic PCR product.

    Techniques Used: CRISPR, In Vitro, Reverse Transcription Polymerase Chain Reaction, Transfection, Allele-specific Oligonucleotide, Sequencing, Derivative Assay, Polymerase Chain Reaction

    20) Product Images from "Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin"

    Article Title: Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007377

    Physical association of the putative helicase (K2ORF4p), mRNA capping enzyme (K2ORF3p), and the large RNAP subunit (K2ORF6p) of the yeast VLEs with VLE-specific DNA. (A) Western blot of HA-K2ORF4p that was affinity-purified from lysates of IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. The strains used are indicated above the lanes. The antibody used is indicated on the left hand side of the strip. The protein detected is indicated on the right hand side of the strip. (B) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K1ORF3 , K2ORF3 ) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. Samples of individually performed gene-specific PCRs were analysed in 2.5% agarose gel stained with ethidium bromide. The identity of the bands (genes) is indicated on the right. M, DNA molecular mass marker (GeneRuler 100 bp Plus DNA Ladder, Fermentas). The respective values are indicated on the left. (C) Western blot of K2ORF3p-HA that was affinity-purified from lysates of IFO1267_pRKL2-14 (K2ORF3p-HA) and IFO1267 (control) cells. (D) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K2ORF3 , K2ORF6 ) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-14 and IFO1267 cells. (E) Western blot of yEGFP3-K2ORF6p that was affinity-purified from lysates of IFO1267_pRKL2-4 (yEGFP3-K2ORF6p) and IFO1267 (control) cells. (F) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K2ORF3 , K2ORF6 ) DNA in chromatin immunoprecipitated using GFP-Trap agarose beads from IFO1267_pRKL2-4 and IFO1267 cells.
    Figure Legend Snippet: Physical association of the putative helicase (K2ORF4p), mRNA capping enzyme (K2ORF3p), and the large RNAP subunit (K2ORF6p) of the yeast VLEs with VLE-specific DNA. (A) Western blot of HA-K2ORF4p that was affinity-purified from lysates of IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. The strains used are indicated above the lanes. The antibody used is indicated on the left hand side of the strip. The protein detected is indicated on the right hand side of the strip. (B) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K1ORF3 , K2ORF3 ) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. Samples of individually performed gene-specific PCRs were analysed in 2.5% agarose gel stained with ethidium bromide. The identity of the bands (genes) is indicated on the right. M, DNA molecular mass marker (GeneRuler 100 bp Plus DNA Ladder, Fermentas). The respective values are indicated on the left. (C) Western blot of K2ORF3p-HA that was affinity-purified from lysates of IFO1267_pRKL2-14 (K2ORF3p-HA) and IFO1267 (control) cells. (D) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K2ORF3 , K2ORF6 ) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-14 and IFO1267 cells. (E) Western blot of yEGFP3-K2ORF6p that was affinity-purified from lysates of IFO1267_pRKL2-4 (yEGFP3-K2ORF6p) and IFO1267 (control) cells. (F) PCR analysis of the presence of chromosomal ( ACT , HGT1 ) or VLE ( K2ORF3 , K2ORF6 ) DNA in chromatin immunoprecipitated using GFP-Trap agarose beads from IFO1267_pRKL2-4 and IFO1267 cells.

    Techniques Used: Western Blot, Affinity Purification, Stripping Membranes, Polymerase Chain Reaction, Activated Clotting Time Assay, Immunoprecipitation, Agarose Gel Electrophoresis, Staining, Marker

    21) Product Images from "Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts"

    Article Title: Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-015-0151-8

    TGFβ1 increased H3K4me1/3 levels on TGFBIp and ECM-associated gene promoters in wild-type and GCD2-homozygous cells. a , b Bar graphs showing H3K4me3 ( a ) and H3K4me1 ( b ) levels at the indicated gene promoters in control and TGFβ1 (5 ng/ml)-stimulated wild-type and GCD2-homozygous corneal fibroblasts. ChIP assays were performed with H3K4me3 and H3K4me1 antibodies. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are shown as the mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; ** P
    Figure Legend Snippet: TGFβ1 increased H3K4me1/3 levels on TGFBIp and ECM-associated gene promoters in wild-type and GCD2-homozygous cells. a , b Bar graphs showing H3K4me3 ( a ) and H3K4me1 ( b ) levels at the indicated gene promoters in control and TGFβ1 (5 ng/ml)-stimulated wild-type and GCD2-homozygous corneal fibroblasts. ChIP assays were performed with H3K4me3 and H3K4me1 antibodies. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are shown as the mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; ** P

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    MLL1 knockdown attenuated TGFβ1-induced increases in H3K4me3 on the promoters of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGF-β1 (5 ng/ml) for 8 h, and H3K4me3 levels at the indicated gene promoters were analyzed. ChIP assays were performed with H3K4me3 antibody. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are presented as mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; ** P
    Figure Legend Snippet: MLL1 knockdown attenuated TGFβ1-induced increases in H3K4me3 on the promoters of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGF-β1 (5 ng/ml) for 8 h, and H3K4me3 levels at the indicated gene promoters were analyzed. ChIP assays were performed with H3K4me3 antibody. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are presented as mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; ** P

    Techniques Used: Infection, shRNA, Selection, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    TGFBIp transcription and H3K4me3 levels in wild-type and GCD2-homozygous corneal fibroblasts. a , b Slit-lamp photographs and pedigree of wild-type, GCD2 heterozygous, and GCD2 homozygous cells. c , d The mRNA and protein levels of TGFBIp in normal and GCD2 corneal fibroblasts was determined by RT-qPCR ( c ) and western blot ( d ). Gene expression was normalized to internal control GAPDH gene, and results are expressed as fold stimulation over control. e , f H3K4me3 and H3K27me3 levels at the Smad binding elements on TGFBIp gene promoters and at the TSS of TGFBIp gene in wild-type and GCD2-homozygous corneal fibroblasts. ChIP assays were performed with H3K4me3 and H3K27me3 antibodies. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the TGFBIp gene promoter to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are the mean fold change relative to the input ± standard error (SE) of enrichment. (mean ± SE; ** P
    Figure Legend Snippet: TGFBIp transcription and H3K4me3 levels in wild-type and GCD2-homozygous corneal fibroblasts. a , b Slit-lamp photographs and pedigree of wild-type, GCD2 heterozygous, and GCD2 homozygous cells. c , d The mRNA and protein levels of TGFBIp in normal and GCD2 corneal fibroblasts was determined by RT-qPCR ( c ) and western blot ( d ). Gene expression was normalized to internal control GAPDH gene, and results are expressed as fold stimulation over control. e , f H3K4me3 and H3K27me3 levels at the Smad binding elements on TGFBIp gene promoters and at the TSS of TGFBIp gene in wild-type and GCD2-homozygous corneal fibroblasts. ChIP assays were performed with H3K4me3 and H3K27me3 antibodies. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the TGFBIp gene promoter to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are the mean fold change relative to the input ± standard error (SE) of enrichment. (mean ± SE; ** P

    Techniques Used: Quantitative RT-PCR, Western Blot, Expressing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Related Articles

    Colorimetric Assay:

    Article Title: Cell-free production of transducible transcription factors for nuclear reprogramming
    Article Snippet: .. The DNA-binding activities of the R9-Nanog, R9-Oct3/4, and R9-Sox2 fusion proteins were assayed by colorimetry utilizing the NoShift Transcription Factor Assay Kit (Novagen) according to the manufacturer’s instructions. .. To assess sequence-specific binding activity, 1–2μg of the R9-Nanog fusion protein, 2–10μg of the R9-Oct3/4 fusion protein or 1–4μg of the R9-Sox2 fusion protein were each incubated in 20μL with 0.5 μM of their respective biotinylated consensus dsDNA binding targets.

    Transcription Factor Assay:

    Article Title: Cell-free production of transducible transcription factors for nuclear reprogramming
    Article Snippet: .. The DNA-binding activities of the R9-Nanog, R9-Oct3/4, and R9-Sox2 fusion proteins were assayed by colorimetry utilizing the NoShift Transcription Factor Assay Kit (Novagen) according to the manufacturer’s instructions. .. To assess sequence-specific binding activity, 1–2μg of the R9-Nanog fusion protein, 2–10μg of the R9-Oct3/4 fusion protein or 1–4μg of the R9-Sox2 fusion protein were each incubated in 20μL with 0.5 μM of their respective biotinylated consensus dsDNA binding targets.

    Multiple Displacement Amplification:

    Article Title: Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs)
    Article Snippet: Primary antibodies used included total ERK, total p38, total JNK (all from Cell Signaling Technologies), and GAPDH (Millipore). .. DNA content of MDA BCCs was measured following ethanol fixation/permeabilization and staining with PI (Sigma). .. Cells derived from monolayer or hanging drop cultures were suspended in PBS containing 2% (vol/vol) FBS and then incubated with an antibody to CD90 (BD Biosciences) for 25 min on ice to discriminate MSCs (CD90-bright) from MDA cells (CD90-negative/dim) during analysis.

    Staining:

    Article Title: Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs)
    Article Snippet: Primary antibodies used included total ERK, total p38, total JNK (all from Cell Signaling Technologies), and GAPDH (Millipore). .. DNA content of MDA BCCs was measured following ethanol fixation/permeabilization and staining with PI (Sigma). .. Cells derived from monolayer or hanging drop cultures were suspended in PBS containing 2% (vol/vol) FBS and then incubated with an antibody to CD90 (BD Biosciences) for 25 min on ice to discriminate MSCs (CD90-bright) from MDA cells (CD90-negative/dim) during analysis.

    Article Title: MDA-7 results in downregulation of AKT concomitant with apoptosis and cell cycle arrest in breast cancer cells
    Article Snippet: Propidium iodide (10 μg/ml) was added just before fluorescence-activated cell sorter analysis. .. DNA content was analyzed using propidium iodide staining (Sigma-Aldrich Chemicals), followed by fluorescence-activated cell sorter analysis to identify the progression of cells through the cell cycle. ..

    TUNEL Assay:

    Article Title: Hot water extract of Agaricus blazei Murrill specifically inhibits growth and induces apoptosis in human pancreatic cancer cells
    Article Snippet: The cells were washed with PBS and then spread on MAS-coated glass slides (Matsunami Glass Industry Ltd., Tokyo, Japan). .. The TUNEL assay was performed to identify DNA fragmentation in situ by using a FragEL™ DNA fragmentation Detection Kit, Colorimetric-TdT Enzyme (Calbiochem, San Diego, CA, USA) in accordance with the manufacturer’s protocol. .. Approximately 500 cells from randomly selected fields were observed at 400× magnification, from which the number of TUNEL-positive cells was counted.

    In Situ:

    Article Title: Hot water extract of Agaricus blazei Murrill specifically inhibits growth and induces apoptosis in human pancreatic cancer cells
    Article Snippet: The cells were washed with PBS and then spread on MAS-coated glass slides (Matsunami Glass Industry Ltd., Tokyo, Japan). .. The TUNEL assay was performed to identify DNA fragmentation in situ by using a FragEL™ DNA fragmentation Detection Kit, Colorimetric-TdT Enzyme (Calbiochem, San Diego, CA, USA) in accordance with the manufacturer’s protocol. .. Approximately 500 cells from randomly selected fields were observed at 400× magnification, from which the number of TUNEL-positive cells was counted.

    Fluorescence:

    Article Title: In vitro culture expansion impairs chondrogenic differentiation and the therapeutic effect of mesenchymal stem cells by regulating the unfolded protein response
    Article Snippet: The GAG content was measured using the dimethylmethylene blue dye binding assay and the absorbance was measured at 525 nm using a microplate reader (Thermo, Karlsruhe, Germany). .. The cellularity was measured based on the DNA content using Hoechst 33258 (Sigma) and the fluorescence intensity was measured at 460 nm using a microplate fluorescence reader (FLX800, Bio-tec, Burlington, Vermont, USA). ..

    Article Title: MDA-7 results in downregulation of AKT concomitant with apoptosis and cell cycle arrest in breast cancer cells
    Article Snippet: Propidium iodide (10 μg/ml) was added just before fluorescence-activated cell sorter analysis. .. DNA content was analyzed using propidium iodide staining (Sigma-Aldrich Chemicals), followed by fluorescence-activated cell sorter analysis to identify the progression of cells through the cell cycle. ..

    ALP Assay:

    Article Title: Aspartic and Glutamic Acid Templated Peptides Conjugation on Plasma Modified Nanofibers for Osteogenic Differentiation of Human Mesenchymal Stem Cells: A Comparative Study
    Article Snippet: To observe initial cellular concentration on scaffolds, DNA content was also measured at day 0. .. Double-stranded DNA content, ALP activity and calcium content of the samples were measured with DNA Quantification Kit (Sigma Aldrich, St. Louis, MO, USA), QuantiChrom ALP assay (Bioassay Systems, Hayward, CA, USA) and QuantiChrom Calcium Assay (Bioassay Systems, Hayward, CA, USA), respectively according to manufacturer’s instructions as previously described . ..

    Activity Assay:

    Article Title: Aspartic and Glutamic Acid Templated Peptides Conjugation on Plasma Modified Nanofibers for Osteogenic Differentiation of Human Mesenchymal Stem Cells: A Comparative Study
    Article Snippet: To observe initial cellular concentration on scaffolds, DNA content was also measured at day 0. .. Double-stranded DNA content, ALP activity and calcium content of the samples were measured with DNA Quantification Kit (Sigma Aldrich, St. Louis, MO, USA), QuantiChrom ALP assay (Bioassay Systems, Hayward, CA, USA) and QuantiChrom Calcium Assay (Bioassay Systems, Hayward, CA, USA), respectively according to manufacturer’s instructions as previously described . ..

    Calcium Assay:

    Article Title: Aspartic and Glutamic Acid Templated Peptides Conjugation on Plasma Modified Nanofibers for Osteogenic Differentiation of Human Mesenchymal Stem Cells: A Comparative Study
    Article Snippet: To observe initial cellular concentration on scaffolds, DNA content was also measured at day 0. .. Double-stranded DNA content, ALP activity and calcium content of the samples were measured with DNA Quantification Kit (Sigma Aldrich, St. Louis, MO, USA), QuantiChrom ALP assay (Bioassay Systems, Hayward, CA, USA) and QuantiChrom Calcium Assay (Bioassay Systems, Hayward, CA, USA), respectively according to manufacturer’s instructions as previously described . ..

    Polymerase Chain Reaction:

    Article Title: Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation
    Article Snippet: Hydroxylamine mutagenesis , spontaneous mutagenesis, and SDM of the gp17 gene was carried out as described in . .. The double-stranded linear DNAs used in this study are 5-kb-length Pl16 DNA linearized by digesting it at the unique PstI site , a 280 bp gene20 PCR product, and a 70 bp DNA prepared using oligonucleotides synthesized from Sigma. .. In all of the experiments, the estimated staining ratio was roughly 1/20–1/5 dye molecules per DNA base pair.

    Synthesized:

    Article Title: Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation
    Article Snippet: Hydroxylamine mutagenesis , spontaneous mutagenesis, and SDM of the gp17 gene was carried out as described in . .. The double-stranded linear DNAs used in this study are 5-kb-length Pl16 DNA linearized by digesting it at the unique PstI site , a 280 bp gene20 PCR product, and a 70 bp DNA prepared using oligonucleotides synthesized from Sigma. .. In all of the experiments, the estimated staining ratio was roughly 1/20–1/5 dye molecules per DNA base pair.

    Real-time Polymerase Chain Reaction:

    Article Title: The Novel Anticytomegalovirus Compound AIC246 (Letermovir) Inhibits Human Cytomegalovirus Replication through a Specific Antiviral Mechanism That Involves the Viral Terminase ▿
    Article Snippet: After the 96-h incubation period, DNA was extracted from infected cells using the blood and cell culture DNA kit (Qiagen, Hilden, Germany). .. Viral DNA was quantified by real-time PCR or slot-blot analysis, and comparable amounts of HCMV DNA were digested with 20 U of KpnI for 2 h, size fractionated on a 0.7% agarose gel by electrophoresis, and blotted to nylon membranes (Millipore, Schwalbach, Germany) via Southern transfer. .. After UV cross-linking, Southern blot analysis was performed according to standard protocols.

    Dot Blot:

    Article Title: The Novel Anticytomegalovirus Compound AIC246 (Letermovir) Inhibits Human Cytomegalovirus Replication through a Specific Antiviral Mechanism That Involves the Viral Terminase ▿
    Article Snippet: After the 96-h incubation period, DNA was extracted from infected cells using the blood and cell culture DNA kit (Qiagen, Hilden, Germany). .. Viral DNA was quantified by real-time PCR or slot-blot analysis, and comparable amounts of HCMV DNA were digested with 20 U of KpnI for 2 h, size fractionated on a 0.7% agarose gel by electrophoresis, and blotted to nylon membranes (Millipore, Schwalbach, Germany) via Southern transfer. .. After UV cross-linking, Southern blot analysis was performed according to standard protocols.

    Agarose Gel Electrophoresis:

    Article Title: The Novel Anticytomegalovirus Compound AIC246 (Letermovir) Inhibits Human Cytomegalovirus Replication through a Specific Antiviral Mechanism That Involves the Viral Terminase ▿
    Article Snippet: After the 96-h incubation period, DNA was extracted from infected cells using the blood and cell culture DNA kit (Qiagen, Hilden, Germany). .. Viral DNA was quantified by real-time PCR or slot-blot analysis, and comparable amounts of HCMV DNA were digested with 20 U of KpnI for 2 h, size fractionated on a 0.7% agarose gel by electrophoresis, and blotted to nylon membranes (Millipore, Schwalbach, Germany) via Southern transfer. .. After UV cross-linking, Southern blot analysis was performed according to standard protocols.

    Electrophoresis:

    Article Title: The Novel Anticytomegalovirus Compound AIC246 (Letermovir) Inhibits Human Cytomegalovirus Replication through a Specific Antiviral Mechanism That Involves the Viral Terminase ▿
    Article Snippet: After the 96-h incubation period, DNA was extracted from infected cells using the blood and cell culture DNA kit (Qiagen, Hilden, Germany). .. Viral DNA was quantified by real-time PCR or slot-blot analysis, and comparable amounts of HCMV DNA were digested with 20 U of KpnI for 2 h, size fractionated on a 0.7% agarose gel by electrophoresis, and blotted to nylon membranes (Millipore, Schwalbach, Germany) via Southern transfer. .. After UV cross-linking, Southern blot analysis was performed according to standard protocols.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore input dna
    Active centromere chromatin displays the signature of elongating RNA polymerase. ( A ) Schematic of the HAC, derived from Nakano et al (2008) , indicating the synthetic alphoid tetO array (green: tetO; white: CENP-B box) and the HAC vector with YAC and BAC cassettes and the blasticidin (Bsr) resistance marker. The region of the alphoid tetO array analysed by ChIP is indicated by green line. ( B ) ChIP analysis in AB2.2.18.21 cells using antibodies of the indicated reactivity. The synthetic (alphoid tetO ) centromere, endogenous chromosome 21 α21-I satellite <t>DNA</t> (alphoid chr.21 ), the 5S rDNA loci and the Bsr gene on the HAC vector were assessed. Data represent the mean and s.d. of three independent ChIP experiments. Note the different scaling of individual panels reflecting different efficiencies of individual antibodies. ( C ) Real-time <t>RT–PCR</t> analysis of synthetic HAC centromere (alphoid tetO ), actively transcribed Bsr marker and endogenous chromosome 21 satellite (alphoid chr.21 ). Expression data are normalized to the copy number of the genomic regions and β-actin mRNA levels (see Materials and methods) and displayed as arbitrary numbers. Data represent the mean and s.e.m. of three independent experiments. Note the log scale.
    Input Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/input dna/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    input dna - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Active centromere chromatin displays the signature of elongating RNA polymerase. ( A ) Schematic of the HAC, derived from Nakano et al (2008) , indicating the synthetic alphoid tetO array (green: tetO; white: CENP-B box) and the HAC vector with YAC and BAC cassettes and the blasticidin (Bsr) resistance marker. The region of the alphoid tetO array analysed by ChIP is indicated by green line. ( B ) ChIP analysis in AB2.2.18.21 cells using antibodies of the indicated reactivity. The synthetic (alphoid tetO ) centromere, endogenous chromosome 21 α21-I satellite DNA (alphoid chr.21 ), the 5S rDNA loci and the Bsr gene on the HAC vector were assessed. Data represent the mean and s.d. of three independent ChIP experiments. Note the different scaling of individual panels reflecting different efficiencies of individual antibodies. ( C ) Real-time RT–PCR analysis of synthetic HAC centromere (alphoid tetO ), actively transcribed Bsr marker and endogenous chromosome 21 satellite (alphoid chr.21 ). Expression data are normalized to the copy number of the genomic regions and β-actin mRNA levels (see Materials and methods) and displayed as arbitrary numbers. Data represent the mean and s.e.m. of three independent experiments. Note the log scale.

    Journal: The EMBO Journal

    Article Title: Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

    doi: 10.1038/emboj.2010.329

    Figure Lengend Snippet: Active centromere chromatin displays the signature of elongating RNA polymerase. ( A ) Schematic of the HAC, derived from Nakano et al (2008) , indicating the synthetic alphoid tetO array (green: tetO; white: CENP-B box) and the HAC vector with YAC and BAC cassettes and the blasticidin (Bsr) resistance marker. The region of the alphoid tetO array analysed by ChIP is indicated by green line. ( B ) ChIP analysis in AB2.2.18.21 cells using antibodies of the indicated reactivity. The synthetic (alphoid tetO ) centromere, endogenous chromosome 21 α21-I satellite DNA (alphoid chr.21 ), the 5S rDNA loci and the Bsr gene on the HAC vector were assessed. Data represent the mean and s.d. of three independent ChIP experiments. Note the different scaling of individual panels reflecting different efficiencies of individual antibodies. ( C ) Real-time RT–PCR analysis of synthetic HAC centromere (alphoid tetO ), actively transcribed Bsr marker and endogenous chromosome 21 satellite (alphoid chr.21 ). Expression data are normalized to the copy number of the genomic regions and β-actin mRNA levels (see Materials and methods) and displayed as arbitrary numbers. Data represent the mean and s.e.m. of three independent experiments. Note the log scale.

    Article Snippet: ChIP'ed and input DNA was subject to real-time PCR analysis using a SYBR Green Mastermix (Sigma) on a LightCycler480 system (Roche).

    Techniques: HAC Assay, Derivative Assay, Plasmid Preparation, BAC Assay, Marker, Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing

    Comparison of methylation data between MeDIP/NimbleGen Promoter + CpGi Array and Sequenom MassArray: Stk31 CpGi promoter region . The Stk31 gene region with a CpGi promoter region that is methylated in the liver, but not in ES or testis cells. The box indicates the position of the methylation peak in the liver that overlaps with the CpGi promoter region analyzed by Sequenom. In the epigram of Sequenom methylation analysis shown at the bottom panel, the blue circles indicate 100% methylation, green circles indicate around 50%methylation and yellow circles indicate 0% methylation. The results from biological duplicate samples are shown. The bottom lane (Sequenom Methylation Analysis) shows the results of analysis of testis DNA after in vitro methylation using M. Sss1 methylase (New England Biolabs).

    Journal: BMC Genomics

    Article Title: Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development

    doi: 10.1186/1471-2164-12-231

    Figure Lengend Snippet: Comparison of methylation data between MeDIP/NimbleGen Promoter + CpGi Array and Sequenom MassArray: Stk31 CpGi promoter region . The Stk31 gene region with a CpGi promoter region that is methylated in the liver, but not in ES or testis cells. The box indicates the position of the methylation peak in the liver that overlaps with the CpGi promoter region analyzed by Sequenom. In the epigram of Sequenom methylation analysis shown at the bottom panel, the blue circles indicate 100% methylation, green circles indicate around 50%methylation and yellow circles indicate 0% methylation. The results from biological duplicate samples are shown. The bottom lane (Sequenom Methylation Analysis) shows the results of analysis of testis DNA after in vitro methylation using M. Sss1 methylase (New England Biolabs).

    Article Snippet: After validating the enrichment of MeDIP DNA, MeDIP DNA and control input DNA were amplified by whole-genome amplification kit (Sigma Aldrich), followed by purification (QIAGEN Quick PCR Purification Kit) and then sent to NimbleGen for Microarray hybridization according to their standard protocol for the array.

    Techniques: Methylation, Methylated DNA Immunoprecipitation, In Vitro

    Assessment of the specificity of the ChIP protocol. A) Arx immunoprecipitation from N2a cells transfected with an Arx-expressing vector or a vector alone and using the Arx C-14 antibody from Santa Cruz. Detection using the anti-Arx-HD antibody revealed a major band at approximately 80 kD, which corresponds to the size of Arx protein. TE: total extracts, IP: immunoprecipitation, Ig: immunoglobulins. B) Enrichment of Ebf3 , Lmo1 and Shox2 promoter regions was assessed by qPCR using either Arx or PolII-immunoprecipitates or using no antibody and was compared to the total input. Gapdh was used as a positive control with DNA Pol II. Here, we show an example of the results obtained in a ChIP experiment with Arx C-14 antibody from Santa Cruz. The enrichment of Ebf3 , Lmo1 and Shox2 was checked similarly for each replicate experiment before DNA was applied to the microarrays.

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: Assessment of the specificity of the ChIP protocol. A) Arx immunoprecipitation from N2a cells transfected with an Arx-expressing vector or a vector alone and using the Arx C-14 antibody from Santa Cruz. Detection using the anti-Arx-HD antibody revealed a major band at approximately 80 kD, which corresponds to the size of Arx protein. TE: total extracts, IP: immunoprecipitation, Ig: immunoglobulins. B) Enrichment of Ebf3 , Lmo1 and Shox2 promoter regions was assessed by qPCR using either Arx or PolII-immunoprecipitates or using no antibody and was compared to the total input. Gapdh was used as a positive control with DNA Pol II. Here, we show an example of the results obtained in a ChIP experiment with Arx C-14 antibody from Santa Cruz. The enrichment of Ebf3 , Lmo1 and Shox2 was checked similarly for each replicate experiment before DNA was applied to the microarrays.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Positive Control

    ChIP-chip results obtained from mouse embryonic brain. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Example of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. Contrarily to N2a cells, in which several continuous probes were often found enriched, only one or two probes were found enriched per gene. C) Venn diagram illustrating the overlap (black) between Arx-immunoprecipitated genes in transfected N2a cells (blue) and mouse embryonic brain (red), and the number of genes with at least 75% match to Arx-binding motif.

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: ChIP-chip results obtained from mouse embryonic brain. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Example of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. Contrarily to N2a cells, in which several continuous probes were often found enriched, only one or two probes were found enriched per gene. C) Venn diagram illustrating the overlap (black) between Arx-immunoprecipitated genes in transfected N2a cells (blue) and mouse embryonic brain (red), and the number of genes with at least 75% match to Arx-binding motif.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Binding Assay

    ChIP-chip results obtained from Arx-transfected N2a cells. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Examples of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics software. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. C) The resulting weighted matrix discovered through the MDModule analysis (top) appears to be similar to the motif identified by Berger et al. [27] (bottom). D) Frequency distribution of scores. The TAATTA motif identified by MDModule was significantly more present in ChIP-enriched sequences (red curve) by comparison to negative control sequences (blue curve).

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: ChIP-chip results obtained from Arx-transfected N2a cells. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Examples of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics software. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. C) The resulting weighted matrix discovered through the MDModule analysis (top) appears to be similar to the motif identified by Berger et al. [27] (bottom). D) Frequency distribution of scores. The TAATTA motif identified by MDModule was significantly more present in ChIP-enriched sequences (red curve) by comparison to negative control sequences (blue curve).

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Chromatin Immunoprecipitation, Transfection, Immunoprecipitation, Software, Negative Control

    Confirmation of Arx binding to candidate promoter regions. A) Arx-immunoprecipitated DNA was compared to input DNA by ChIP/QFM-PCR to determine ChIP enrichment of 21 putative target genes. No enrichment occurred for the Vapb promoter which was consistently negative in all ChIP experiments. Bar heights represent log 10 enrichment of the signal obtained for Arx-immunoprecipitated DNA versus input DNA for each promoter using site-specific primers. The arrows above the bars indicate sequences in which the previously defined Arx-binding motif was found. B) Confirmation of Arx binding to 5 different promoter sequences identified by ChIP using a luciferase reporter gene assay. Firefly luciferase data were normalized to Renilla luciferase expression and data are presented as the percentage of transcriptional activity compared to the vector control. Arx regulation was confirmed for Lmo1 , Lmo3 , Sh3tc2 , Calb2 and Cdh2 promoter regions as transcriptional activity was repressed in the presence of Arx by comparison to the control transfection, whereas it had no effect on the plasmid alone pGL4.23. Error bars indicate SEM.

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: Confirmation of Arx binding to candidate promoter regions. A) Arx-immunoprecipitated DNA was compared to input DNA by ChIP/QFM-PCR to determine ChIP enrichment of 21 putative target genes. No enrichment occurred for the Vapb promoter which was consistently negative in all ChIP experiments. Bar heights represent log 10 enrichment of the signal obtained for Arx-immunoprecipitated DNA versus input DNA for each promoter using site-specific primers. The arrows above the bars indicate sequences in which the previously defined Arx-binding motif was found. B) Confirmation of Arx binding to 5 different promoter sequences identified by ChIP using a luciferase reporter gene assay. Firefly luciferase data were normalized to Renilla luciferase expression and data are presented as the percentage of transcriptional activity compared to the vector control. Arx regulation was confirmed for Lmo1 , Lmo3 , Sh3tc2 , Calb2 and Cdh2 promoter regions as transcriptional activity was repressed in the presence of Arx by comparison to the control transfection, whereas it had no effect on the plasmid alone pGL4.23. Error bars indicate SEM.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Binding Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Luciferase, Reporter Gene Assay, Expressing, Activity Assay, Plasmid Preparation, Transfection

    FMR1 locus (h)MeDIP profiling identifies novel regions of 5mC and 5hmC changes in FXS patient PBMC samples. a . Experimental overview. Methylated DNA immunoprecipitation (MeDIP) assay was used to profile DNA methylation (5mC), DNA hydroxymethylation (5hmC). Chromatin immunoprecipitation (ChIP) was used to profile histone post-translational modifications (PTM). Antibody (Ab) b – c . The graphs illustrate the relative enrichment of the indicated mark (log2 fold enrichment) in FXS ( red ) and control ( blue ) PBMCs. The upper panel illustrates the chromosomal and genomic location locations as well as the indicated referenced Refseq genes: FMR1 , and referenced antisense non coding RNAs ( FMR1AS1 and L29074.3). This snapshot illustrates the epigenetic landscape over a region covering 79 kb on ChrX: 146971000-147050000 ( c ). RPL19 and GAPDH provide controls regions of no changes in 5mC and 5hmC ( b ). In the graphs, the thicker lines indicate higher deviation between biological replicates (controls n = 4; FXS n = 8) (aggregation mean, sliding window 529 bp). Regions I to V were selected as regions of strongest apparent epigenetic variation across epigenetic marks and cell types based on epigenomic landscape visualization illustrated in Figs. 1 and 3 . The chromosome coordinates of these regions are provided in Table 1

    Journal: Clinical Epigenetics

    Article Title: Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome

    doi: 10.1186/s13148-016-0181-x

    Figure Lengend Snippet: FMR1 locus (h)MeDIP profiling identifies novel regions of 5mC and 5hmC changes in FXS patient PBMC samples. a . Experimental overview. Methylated DNA immunoprecipitation (MeDIP) assay was used to profile DNA methylation (5mC), DNA hydroxymethylation (5hmC). Chromatin immunoprecipitation (ChIP) was used to profile histone post-translational modifications (PTM). Antibody (Ab) b – c . The graphs illustrate the relative enrichment of the indicated mark (log2 fold enrichment) in FXS ( red ) and control ( blue ) PBMCs. The upper panel illustrates the chromosomal and genomic location locations as well as the indicated referenced Refseq genes: FMR1 , and referenced antisense non coding RNAs ( FMR1AS1 and L29074.3). This snapshot illustrates the epigenetic landscape over a region covering 79 kb on ChrX: 146971000-147050000 ( c ). RPL19 and GAPDH provide controls regions of no changes in 5mC and 5hmC ( b ). In the graphs, the thicker lines indicate higher deviation between biological replicates (controls n = 4; FXS n = 8) (aggregation mean, sliding window 529 bp). Regions I to V were selected as regions of strongest apparent epigenetic variation across epigenetic marks and cell types based on epigenomic landscape visualization illustrated in Figs. 1 and 3 . The chromosome coordinates of these regions are provided in Table 1

    Article Snippet: For microarray analysis, 50 ng of input DNA and 1/2 (h)MeDIP-enriched DNA was amplified using WGA2: GenomePlex Complete Whole Genome Amplification kit (Sigma).

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