mcrbc  (New England Biolabs)


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    McrBC
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    McrBC 2 500 units
    Catalog Number:
    m0272l
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    290
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    New England Biolabs mcrbc
    McrBC
    McrBC 2 500 units
    https://www.bioz.com/result/mcrbc/product/New England Biolabs
    Average 99 stars, based on 93 article reviews
    Price from $9.99 to $1999.99
    mcrbc - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation"

    Article Title: SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv958

    The suvh1–1 mutation does not affect DNA methylation. ( A and B ) McrBC-qPCR analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.
    Figure Legend Snippet: The suvh1–1 mutation does not affect DNA methylation. ( A and B ) McrBC-qPCR analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.

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

    2) Product Images from "Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential"

    Article Title: Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2545-1

    qPCR validation of microarray results for remarkable genes showing significant changes in DNA methylation and transcript levels between d1 and d7 in neonatal murine hearts. The left panels show DNA methylation and gene expression microarray results represented by red bars and black markers, respectively; DNA methylation is expressed as MeDIP enrichment and gene expression levels as normalized microarray signals. The black markers preceding that indicating the neonatal d1 represent gene expression levels in embryonic hearts E16, E18, E19, E20. The middle panels present CpG methylation estimated with Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) where the DNA methylation levels correspond to the amounts of DNA undigested by CpG methylation dependent McrBC enzyme (1-(McrBC/Input). The right panels demonstrate transcript levels determined with qPCR as the ratios to the reference transcript of the Tbp gene (TATA binding protein gene). The microarray results were determined for pooled samples of 3 mice. The qPCR results represent average values obtained for three individuals for each developmental time-point. The statistical significance has been determined with two-tailed heteroscedastic Student’s t -test. The complete results of qPCR validation are collected in Additional file 2 : F2
    Figure Legend Snippet: qPCR validation of microarray results for remarkable genes showing significant changes in DNA methylation and transcript levels between d1 and d7 in neonatal murine hearts. The left panels show DNA methylation and gene expression microarray results represented by red bars and black markers, respectively; DNA methylation is expressed as MeDIP enrichment and gene expression levels as normalized microarray signals. The black markers preceding that indicating the neonatal d1 represent gene expression levels in embryonic hearts E16, E18, E19, E20. The middle panels present CpG methylation estimated with Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) where the DNA methylation levels correspond to the amounts of DNA undigested by CpG methylation dependent McrBC enzyme (1-(McrBC/Input). The right panels demonstrate transcript levels determined with qPCR as the ratios to the reference transcript of the Tbp gene (TATA binding protein gene). The microarray results were determined for pooled samples of 3 mice. The qPCR results represent average values obtained for three individuals for each developmental time-point. The statistical significance has been determined with two-tailed heteroscedastic Student’s t -test. The complete results of qPCR validation are collected in Additional file 2 : F2

    Techniques Used: Real-time Polymerase Chain Reaction, Microarray, DNA Methylation Assay, Expressing, Methylated DNA Immunoprecipitation, CpG Methylation Assay, Methylation, Binding Assay, Mouse Assay, Two Tailed Test

    3) Product Images from "High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening"

    Article Title: High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02782-9

    Differential ripening-related demethylation of the promoter of RIN between pericarp and locular tissue. a RIN self-binding sites and differentially methylated regions in the RIN ). ChIP-seq plot depicts position of RIN binding, with peak height representing the number of reads covering each site (i.e., read depth). Red bars indicate cytosine methylation ratios at the equivalent position. Two promoter fragments analyzed by McrBC-PCR are indicated by black bars. b McrBC-PCR analysis of two RIN promoter fragments in pericarp (Pe) and locular tissue (Lo) at four stages spanning fruit expansion and ripening. A total of 0.5 μg genomic DNA was digested by McrBC (NEB) with GTP (+), or without GTP (−) as a negative control. An unmethylated region of the PME (Solyc03g123630) promoter is included as a positive control, and a highly methylated region of the CNR (Solyc02g077920) promoter showing complete digestion of methylated DNA. DPA, days post-anthesis; MG, mature green; Br, breaker; Pk, pink; RR, red ripe)
    Figure Legend Snippet: Differential ripening-related demethylation of the promoter of RIN between pericarp and locular tissue. a RIN self-binding sites and differentially methylated regions in the RIN ). ChIP-seq plot depicts position of RIN binding, with peak height representing the number of reads covering each site (i.e., read depth). Red bars indicate cytosine methylation ratios at the equivalent position. Two promoter fragments analyzed by McrBC-PCR are indicated by black bars. b McrBC-PCR analysis of two RIN promoter fragments in pericarp (Pe) and locular tissue (Lo) at four stages spanning fruit expansion and ripening. A total of 0.5 μg genomic DNA was digested by McrBC (NEB) with GTP (+), or without GTP (−) as a negative control. An unmethylated region of the PME (Solyc03g123630) promoter is included as a positive control, and a highly methylated region of the CNR (Solyc02g077920) promoter showing complete digestion of methylated DNA. DPA, days post-anthesis; MG, mature green; Br, breaker; Pk, pink; RR, red ripe)

    Techniques Used: Binding Assay, Methylation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Positive Control

    4) Product Images from "PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit"

    Article Title: PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit

    Journal: Horticulture Research

    doi: 10.1038/s41438-019-0220-9

    Methylation levels of the PbGA2ox8 promoter regions among “Red Zaosu”, “Zaosu”, and their progeny as assessed by McrBc-PCR. The 1300-bp region upstream of the ATG translational start site of PbGA2ox8 was divided into four overlapping fragments: promoter region 1 ( pG28-1 ), promoter region 2 ( pG28-2 ), promoter region 3 ( pG28-3 ), and promoter region 4 ( pG28-4 ). In total, 0.5 μg of genomic DNA was digested with McrBc in the presence GTP (+). Samples lacking GTP served as the negative controls (−). F1R and F1G, pooled red-, and green-leafed segregants, respectively, from the 15F1 population; F1R1–11 and F1G1–11, 11 randomly selected red and green-leafed individuals, respectively, from the 15F1 population; +1, ATG translational start site.
    Figure Legend Snippet: Methylation levels of the PbGA2ox8 promoter regions among “Red Zaosu”, “Zaosu”, and their progeny as assessed by McrBc-PCR. The 1300-bp region upstream of the ATG translational start site of PbGA2ox8 was divided into four overlapping fragments: promoter region 1 ( pG28-1 ), promoter region 2 ( pG28-2 ), promoter region 3 ( pG28-3 ), and promoter region 4 ( pG28-4 ). In total, 0.5 μg of genomic DNA was digested with McrBc in the presence GTP (+). Samples lacking GTP served as the negative controls (−). F1R and F1G, pooled red-, and green-leafed segregants, respectively, from the 15F1 population; F1R1–11 and F1G1–11, 11 randomly selected red and green-leafed individuals, respectively, from the 15F1 population; +1, ATG translational start site.

    Techniques Used: Methylation, Polymerase Chain Reaction

    5) Product Images from "Chromatin and sequence features that define the fine and gross structure of genomic methylation patterns"

    Article Title: Chromatin and sequence features that define the fine and gross structure of genomic methylation patterns

    Journal: Genome Research

    doi: 10.1101/gr.101535.109

    High-resolution genome-wide methylation profiling and genome-wide DNA methylation trends. ( A ) Browser view of Methyl-MAPS data from the genomic region spanning the BIK gene. Individual mapped sequence reads are shown in the top raw data tracks. Red sequences were resistant to methylation-sensitive restriction endonucleases (RE) and are therefore methylated. Blue sequences were resistant to the methylation-dependent McrBC complex and are unmethylated. Tick marks in both tracks along the top of the figure and within each sequence indicate locations of individual RE and McrBC recognition sequences. Methylation data is also presented in a concise view, where each CpG is assigned a methylation score from the ratio of methylated to total (unmethylated and methylated) sequences covering each CpG site. The bulk of the BIK gene is methylated, while the CpG-rich promoter is unmethylated. ( B ) Methylation of the SVA retrotransposon in a repeat-rich region of chr 19. While the CpG density is comparable to that of the CpG island of the BIK gene shown in A , the SVA retrotransposon is densely methylated.
    Figure Legend Snippet: High-resolution genome-wide methylation profiling and genome-wide DNA methylation trends. ( A ) Browser view of Methyl-MAPS data from the genomic region spanning the BIK gene. Individual mapped sequence reads are shown in the top raw data tracks. Red sequences were resistant to methylation-sensitive restriction endonucleases (RE) and are therefore methylated. Blue sequences were resistant to the methylation-dependent McrBC complex and are unmethylated. Tick marks in both tracks along the top of the figure and within each sequence indicate locations of individual RE and McrBC recognition sequences. Methylation data is also presented in a concise view, where each CpG is assigned a methylation score from the ratio of methylated to total (unmethylated and methylated) sequences covering each CpG site. The bulk of the BIK gene is methylated, while the CpG-rich promoter is unmethylated. ( B ) Methylation of the SVA retrotransposon in a repeat-rich region of chr 19. While the CpG density is comparable to that of the CpG island of the BIK gene shown in A , the SVA retrotransposon is densely methylated.

    Techniques Used: Genome Wide, Methylation, DNA Methylation Assay, Sequencing

    6) Product Images from "Exendin-4 promotes extracellular-superoxide dismutase expression in A549 cells through DNA demethylation"

    Article Title: Exendin-4 promotes extracellular-superoxide dismutase expression in A549 cells through DNA demethylation

    Journal: Journal of Clinical Biochemistry and Nutrition

    doi: 10.3164/jcbn.15-16

    Involvement of DNA demethylation in exendin-4-inducible EC-SOD expression in A549 cells. A549 cells were treated with 100 nM exendin-4 or 1 µM Aza for 24 h. (A) After these treatments, genomic DNA from A549 cells was purified, and followed by McrBC digestion-real-time RT-PCR. Data are shown as the mean ± SD ( n = 3). * p
    Figure Legend Snippet: Involvement of DNA demethylation in exendin-4-inducible EC-SOD expression in A549 cells. A549 cells were treated with 100 nM exendin-4 or 1 µM Aza for 24 h. (A) After these treatments, genomic DNA from A549 cells was purified, and followed by McrBC digestion-real-time RT-PCR. Data are shown as the mean ± SD ( n = 3). * p

    Techniques Used: Expressing, Purification, Quantitative RT-PCR

    7) Product Images from "Independent functions of DNMT1 and USP7 at replication foci"

    Article Title: Independent functions of DNMT1 and USP7 at replication foci

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0179-z

    Ablation of USP7 does not affect steady-state level of DNMT1 or global DNA methylation. a MEFs that lack USP7 after Cre-mediated excision of a Floxed allele of Usp7 show normal levels of DNMT1 (lane 2) but no detectable USP7 protein. Biological replicates are shown as Experiments 1 and 2. b Normal expression of DNMT1 and UHRF1 in human H1299 lung carcinoma cells containing an inducible shRNA against USP7 mRNA. Ablation of USP7 has no detectable effect on expression of other proteins. c Normal steady-state expression of USP7 in the absence of DNMT1 in ES cells. d Normal DNA methylation in MEFs that lack USP7. McrBC cleaves methylated DNA; HpaII cleaves unmethylated DNA at CCGG sites; MspI cleaves CCGG sites whether methylated or unmethylated. DNA from wild-type and Usp7 − / − MEFs show similar patterns of resistance to both McrBC and HpaII. e Removal of USP7 by an inducible shRNA against USP7 from H1299 human lung carcinoma cells does not affect DNA methylation levels as assessed by resistance of DNA to HpaII. f LUMA analysis shows that DNA methylation is not measurably affected by removal of USP7. n = 2 (biological replicates). Error bars show standard deviations, center value is mean, and p values were calculated using the two-tailed t test
    Figure Legend Snippet: Ablation of USP7 does not affect steady-state level of DNMT1 or global DNA methylation. a MEFs that lack USP7 after Cre-mediated excision of a Floxed allele of Usp7 show normal levels of DNMT1 (lane 2) but no detectable USP7 protein. Biological replicates are shown as Experiments 1 and 2. b Normal expression of DNMT1 and UHRF1 in human H1299 lung carcinoma cells containing an inducible shRNA against USP7 mRNA. Ablation of USP7 has no detectable effect on expression of other proteins. c Normal steady-state expression of USP7 in the absence of DNMT1 in ES cells. d Normal DNA methylation in MEFs that lack USP7. McrBC cleaves methylated DNA; HpaII cleaves unmethylated DNA at CCGG sites; MspI cleaves CCGG sites whether methylated or unmethylated. DNA from wild-type and Usp7 − / − MEFs show similar patterns of resistance to both McrBC and HpaII. e Removal of USP7 by an inducible shRNA against USP7 from H1299 human lung carcinoma cells does not affect DNA methylation levels as assessed by resistance of DNA to HpaII. f LUMA analysis shows that DNA methylation is not measurably affected by removal of USP7. n = 2 (biological replicates). Error bars show standard deviations, center value is mean, and p values were calculated using the two-tailed t test

    Techniques Used: DNA Methylation Assay, Expressing, shRNA, Methylation, Two Tailed Test

    8) Product Images from "Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs"

    Article Title: Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2011.458

    DNA methylation and expression analysis of H06 locus. ( A ) Methylation of H06 genomic DNA using restriction enzyme McrBC. Completely digested DNA indicates hypermethylation. Error bars indicate the 95% confidence intervals of three replicates.
    Figure Legend Snippet: DNA methylation and expression analysis of H06 locus. ( A ) Methylation of H06 genomic DNA using restriction enzyme McrBC. Completely digested DNA indicates hypermethylation. Error bars indicate the 95% confidence intervals of three replicates.

    Techniques Used: DNA Methylation Assay, Expressing, Methylation

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep01400

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

    Techniques Used: Methylation, Sequencing, Blocking Assay

    10) Product Images from "Genes and Transposons Are Differentially Methylated in Plants, but Not in Mammals"

    Article Title: Genes and Transposons Are Differentially Methylated in Plants, but Not in Mammals

    Journal: Genome Research

    doi: 10.1101/gr.1784803

    Transposons, but not genes, are methylated in maize. Genomic DNA was digested with McrBC during 0 min, 25 min, or 8 h, with or without Sss )
    Figure Legend Snippet: Transposons, but not genes, are methylated in maize. Genomic DNA was digested with McrBC during 0 min, 25 min, or 8 h, with or without Sss )

    Techniques Used: Methylation

    McrPCR. Equal samples of genomic DNA are either pretreated with CpG methylase ( Sss I methylase), or not, and both samples are digested in a time course with McrBC (only time zero and complete digestion are shown). PCR is then performed using specific primers (arrows). If the target sequence (gray box) is methylated in the genome, there will be a decrease in the amount of PCR product following McrBC digestion with or without Sss I methylase pretreatment. If the target is not methylated, a decrease in PCR yield will only be evident following Sss I methylase pretreatment.
    Figure Legend Snippet: McrPCR. Equal samples of genomic DNA are either pretreated with CpG methylase ( Sss I methylase), or not, and both samples are digested in a time course with McrBC (only time zero and complete digestion are shown). PCR is then performed using specific primers (arrows). If the target sequence (gray box) is methylated in the genome, there will be a decrease in the amount of PCR product following McrBC digestion with or without Sss I methylase pretreatment. If the target is not methylated, a decrease in PCR yield will only be evident following Sss I methylase pretreatment.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Methylation

    11) Product Images from "Directed Evolution of Improved Zinc Finger Methyltransferases"

    Article Title: Directed Evolution of Improved Zinc Finger Methyltransferases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096931

    Schematics of the vector, library, proteins, and selection used in these experiments. (A) The vector used in selections. The vector encodes for both heterodimeric fragments fused to zinc fingers under the control of separate inducible arabinose ( pBAD ) and IPTG ( lac ) promoters, a target site, and the araC gene. (B) A schema of the zinc finger-fused, bifurcated M.SssI and the mutagenized codons used in library construction. Codons corresponding to residues 297–301 of M.SssI (located in the C-terminal fragment) were randomized. Numbering scheme is that of the wildtype M.SssI. (C) An assembled zinc finger-fused heterodimeric M.SssI methyltransferase assembled at the target site. The target site consists of an internal CpG site nested within an FspI restriction site, and flanked by HS1 and HS2 recognition sequences. (D) The non-target site used to assess off-target methylation. The non-target site lacks HS1 and HS2 zinc finger binding sites, but contains a CpG site nested within an SnaBI restriction site (E) An overview of the selections used in this experiment. The schematic illustrates the fates of plasmids encoding inactive methyltransferase (digested by FspI, left), a desired targeting methyltransferase methylated at the target site (not digested, middle) and a nonspecific methyltransferase methylating multiple M.SssI (i.e CpG) sites (digested by McrBC, right).
    Figure Legend Snippet: Schematics of the vector, library, proteins, and selection used in these experiments. (A) The vector used in selections. The vector encodes for both heterodimeric fragments fused to zinc fingers under the control of separate inducible arabinose ( pBAD ) and IPTG ( lac ) promoters, a target site, and the araC gene. (B) A schema of the zinc finger-fused, bifurcated M.SssI and the mutagenized codons used in library construction. Codons corresponding to residues 297–301 of M.SssI (located in the C-terminal fragment) were randomized. Numbering scheme is that of the wildtype M.SssI. (C) An assembled zinc finger-fused heterodimeric M.SssI methyltransferase assembled at the target site. The target site consists of an internal CpG site nested within an FspI restriction site, and flanked by HS1 and HS2 recognition sequences. (D) The non-target site used to assess off-target methylation. The non-target site lacks HS1 and HS2 zinc finger binding sites, but contains a CpG site nested within an SnaBI restriction site (E) An overview of the selections used in this experiment. The schematic illustrates the fates of plasmids encoding inactive methyltransferase (digested by FspI, left), a desired targeting methyltransferase methylated at the target site (not digested, middle) and a nonspecific methyltransferase methylating multiple M.SssI (i.e CpG) sites (digested by McrBC, right).

    Techniques Used: Plasmid Preparation, Selection, Zinc-Fingers, Methylation, Binding Assay

    12) Product Images from "HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis 1HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis 1 [W]HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis 1 [W] [OA]"

    Article Title: HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis 1HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis 1 [W]HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis 1 [W] [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.111.184275

    Gene expression (A) and cytosine methylation (B) analyses of HDA6-regulated TEs in met1-3 plants. d, McrBC-digested DNA input; u, undigested DNA input.
    Figure Legend Snippet: Gene expression (A) and cytosine methylation (B) analyses of HDA6-regulated TEs in met1-3 plants. d, McrBC-digested DNA input; u, undigested DNA input.

    Techniques Used: Expressing, Methylation

    13) Product Images from "Tempered mlo broad-spectrum resistance to barley powdery mildew in an Ethiopian landrace"

    Article Title: Tempered mlo broad-spectrum resistance to barley powdery mildew in an Ethiopian landrace

    Journal: Scientific Reports

    doi: 10.1038/srep29558

    DNA methylation status of Mlo in landrace Eth295, cv. Baudin and cv. Westminster. ( A ) DNA methylation status was determined by digestion with McrBC followed by quantitative PCR at the promoter and UTR region of Mlo gene. Undigested genomic DNA was used as a control. Four biological replicates were used for each accession and real time PCR was performed with two McrBC digest replicates and four qPCR technical replicates. Error bars are standard errors and significant differences are determined using the Student’s t-test; *** P
    Figure Legend Snippet: DNA methylation status of Mlo in landrace Eth295, cv. Baudin and cv. Westminster. ( A ) DNA methylation status was determined by digestion with McrBC followed by quantitative PCR at the promoter and UTR region of Mlo gene. Undigested genomic DNA was used as a control. Four biological replicates were used for each accession and real time PCR was performed with two McrBC digest replicates and four qPCR technical replicates. Error bars are standard errors and significant differences are determined using the Student’s t-test; *** P

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

    14) Product Images from "DNA Methylation Affects the SP1-regulated Transcription of FOXF2 in Breast Cancer Cells *"

    Article Title: DNA Methylation Affects the SP1-regulated Transcription of FOXF2 in Breast Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.636126

    DNA methylation represses FOXF2 promoter activity. A , following in vitro methylation with SssI, HhaI, or HpaII methylase, the proximal promoter region (−655/+128) of FOXF2 was digested with McrBC, HhaI, or HpaII to confirm the methylation status.
    Figure Legend Snippet: DNA methylation represses FOXF2 promoter activity. A , following in vitro methylation with SssI, HhaI, or HpaII methylase, the proximal promoter region (−655/+128) of FOXF2 was digested with McrBC, HhaI, or HpaII to confirm the methylation status.

    Techniques Used: DNA Methylation Assay, Activity Assay, In Vitro, Methylation

    15) Product Images from "The Genome of the Stick Insect Medauroidea extradentata Is Strongly Methylated within Genes and Repetitive DNA"

    Article Title: The Genome of the Stick Insect Medauroidea extradentata Is Strongly Methylated within Genes and Repetitive DNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007223

    Differential restriction analysis of Medauroidea extradentata . Equivalent amounts of genomic DNA were digested and analyzed using MspI (Ms), HpaII (Hp), MboI (Mb), Bsp143I (Bs) and McrBC. For comparison, equivalent genomic digestions using McrBC and DNA from Drosophila melanogaster (Dr), Medauroidea extradentata (Me) and Homo sapiens (Ho) are shown. The track labelled L contains GeneRuler® Ladder Mix (Fermentas).
    Figure Legend Snippet: Differential restriction analysis of Medauroidea extradentata . Equivalent amounts of genomic DNA were digested and analyzed using MspI (Ms), HpaII (Hp), MboI (Mb), Bsp143I (Bs) and McrBC. For comparison, equivalent genomic digestions using McrBC and DNA from Drosophila melanogaster (Dr), Medauroidea extradentata (Me) and Homo sapiens (Ho) are shown. The track labelled L contains GeneRuler® Ladder Mix (Fermentas).

    Techniques Used: Mass Spectrometry

    16) Product Images from "Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential"

    Article Title: Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2545-1

    qPCR validation of microarray results for remarkable genes showing significant changes in DNA methylation and transcript levels between d1 and d7 in neonatal murine hearts. The left panels show DNA methylation and gene expression microarray results represented by red bars and black markers, respectively; DNA methylation is expressed as MeDIP enrichment and gene expression levels as normalized microarray signals. The black markers preceding that indicating the neonatal d1 represent gene expression levels in embryonic hearts E16, E18, E19, E20. The middle panels present CpG methylation estimated with Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) where the DNA methylation levels correspond to the amounts of DNA undigested by CpG methylation dependent McrBC enzyme (1-(McrBC/Input). The right panels demonstrate transcript levels determined with qPCR as the ratios to the reference transcript of the Tbp gene (TATA binding protein gene). The microarray results were determined for pooled samples of 3 mice. The qPCR results represent average values obtained for three individuals for each developmental time-point. The statistical significance has been determined with two-tailed heteroscedastic Student’s t -test. The complete results of qPCR validation are collected in Additional file 2 : F2
    Figure Legend Snippet: qPCR validation of microarray results for remarkable genes showing significant changes in DNA methylation and transcript levels between d1 and d7 in neonatal murine hearts. The left panels show DNA methylation and gene expression microarray results represented by red bars and black markers, respectively; DNA methylation is expressed as MeDIP enrichment and gene expression levels as normalized microarray signals. The black markers preceding that indicating the neonatal d1 represent gene expression levels in embryonic hearts E16, E18, E19, E20. The middle panels present CpG methylation estimated with Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) where the DNA methylation levels correspond to the amounts of DNA undigested by CpG methylation dependent McrBC enzyme (1-(McrBC/Input). The right panels demonstrate transcript levels determined with qPCR as the ratios to the reference transcript of the Tbp gene (TATA binding protein gene). The microarray results were determined for pooled samples of 3 mice. The qPCR results represent average values obtained for three individuals for each developmental time-point. The statistical significance has been determined with two-tailed heteroscedastic Student’s t -test. The complete results of qPCR validation are collected in Additional file 2 : F2

    Techniques Used: Real-time Polymerase Chain Reaction, Microarray, DNA Methylation Assay, Expressing, Methylated DNA Immunoprecipitation, CpG Methylation Assay, Methylation, Binding Assay, Mouse Assay, Two Tailed Test

    17) Product Images from "Epigenetic Reprogramming of the Type III Interferon Response Potentiates Antiviral Activity and Suppresses Tumor Growth"

    Article Title: Epigenetic Reprogramming of the Type III Interferon Response Potentiates Antiviral Activity and Suppresses Tumor Growth

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001758

    IFNLR1 promoter methylation negatively correlates with IFN-λ responsiveness. (A) Huh7 and U87 genomic DNA was digested with McrBC in the presence or absence of GTP and used as template for nested PCR with primers specific for the IFNLR1 promoter. (B) Huh7, HepG2, U87, and U373 genomic DNA was subject to bisulfite conversion sequencing. Each circle represents one CpG dinucleotide, with filled circles indicating methylated motifs and open circles nonmethylated motifs. Each row represents an individual clone of the population. Lower numbers indicate relative distance to the TSS. (C) Quantification of the methylation status on both CpG islands in (B). (D) U87 cells were cultured in the presence of vehicle control DMSO, 3 µM, or 10 µM 5azadC for 72 h. IFNLR1 and IL10RB expression was examined by RT-qPCR. In all panels, data represent the mean and standard error of the mean (SEM) of at least three experiments.
    Figure Legend Snippet: IFNLR1 promoter methylation negatively correlates with IFN-λ responsiveness. (A) Huh7 and U87 genomic DNA was digested with McrBC in the presence or absence of GTP and used as template for nested PCR with primers specific for the IFNLR1 promoter. (B) Huh7, HepG2, U87, and U373 genomic DNA was subject to bisulfite conversion sequencing. Each circle represents one CpG dinucleotide, with filled circles indicating methylated motifs and open circles nonmethylated motifs. Each row represents an individual clone of the population. Lower numbers indicate relative distance to the TSS. (C) Quantification of the methylation status on both CpG islands in (B). (D) U87 cells were cultured in the presence of vehicle control DMSO, 3 µM, or 10 µM 5azadC for 72 h. IFNLR1 and IL10RB expression was examined by RT-qPCR. In all panels, data represent the mean and standard error of the mean (SEM) of at least three experiments.

    Techniques Used: Methylation, Nested PCR, Sequencing, Cell Culture, Expressing, Quantitative RT-PCR

    18) Product Images from "Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification"

    Article Title: Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-179

    Comparison of HM-PCR and HM-WGA-PCR results for eleven CGIs . Genomic DNA from peripheral blood leukocytes was digested with Mock (lane 1), Hpa II or Hha I (lane 2), Msp I (lane 3), or McrBC (lane 4). The digested genomic DNA were used for PCR amplification either directly (left; HM-PCR) or after whole-genome-amplification (right; HM-WGA-PCR) using primer pairs for the eight CGIs on chromosome 21 (A~D) and three CGIs on chromosome 11 (E). Here, when Hha I-digested genomic DNA was used in lane 2, 1 ul of distilled water was used in lane 3 in place of Msp I-digested genomic DNA. PCR products were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. Results of bisulfite sequencing are shown for the eight CGIs on chromosome 21 (A~D). Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Each row of circles represents each sequenced clone of bisufite PCR products.
    Figure Legend Snippet: Comparison of HM-PCR and HM-WGA-PCR results for eleven CGIs . Genomic DNA from peripheral blood leukocytes was digested with Mock (lane 1), Hpa II or Hha I (lane 2), Msp I (lane 3), or McrBC (lane 4). The digested genomic DNA were used for PCR amplification either directly (left; HM-PCR) or after whole-genome-amplification (right; HM-WGA-PCR) using primer pairs for the eight CGIs on chromosome 21 (A~D) and three CGIs on chromosome 11 (E). Here, when Hha I-digested genomic DNA was used in lane 2, 1 ul of distilled water was used in lane 3 in place of Msp I-digested genomic DNA. PCR products were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. Results of bisulfite sequencing are shown for the eight CGIs on chromosome 21 (A~D). Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Each row of circles represents each sequenced clone of bisufite PCR products.

    Techniques Used: Polymerase Chain Reaction, Whole Genome Amplification, Amplification, Staining, Methylation Sequencing, Methylation

    Principles of the Hpa II- McrBC whole-genome-amplification PCR (HM-WGA-PCR) method . The two parallel lines in the
    Figure Legend Snippet: Principles of the Hpa II- McrBC whole-genome-amplification PCR (HM-WGA-PCR) method . The two parallel lines in the "Digestion" panel indicate genomic amplicons from both alleles. The circled "m" indicates a methylated CpG dinucleotide. Hpa II digests unmethylated CCGG, but not methylated C m CGG. In contrast, McrBC digests methylated R m C 40-80 R m C sequences, but not unmethylated RC 40-80 RC. Genomic DNA is digested with Hpa II and McrBC independently. Subsequently, an aliquot of each restriction-enzyme-digested DNA (50 ng) is subjected to whole-genome-amplification (WGA) to yield 5 μg of whole-genome-amplified DNA. Using an aliquot of the amplified DNA (50 ng), the target DNA region is PCR-amplified by the primer pair (dotted arrows). The PCR products from the Hpa II/ Hha I-digested and McrBC -digested DNA are electrophoresed, stained with ethidium bromide, and visualized by UV illumination. If an amplicon is fully methylated (i.e., complete methylation), it is digested by McrBC , but not by Hpa II. Thus, it is amplified from the Hpa II-digested and whole-genome-amplified DNA, but not from the McrBC -digested and whole-genome-amplified DNA. By contrast, if an amplicon totally escapes methylation (i.e., null methylation), it is digested by Hpa II, but not by McrBC . Thus, it is amplified from McrBC -digested and whole-genome-amplified DNA, but not from Hpa II-digested and whole-genome-amplified DNA. If an amplicon contains both methylated and unmethylated alleles (i.e., composite methylation), it is amplified from both whole-genome-amplified templates. If an amplicon is partially methylated on both alleles (i.e., incomplete methylation), it is amplified from neither whole-genome-amplified template.

    Techniques Used: Whole Genome Amplification, Polymerase Chain Reaction, Methylation, Amplification, Staining

    19) Product Images from "High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening"

    Article Title: High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02782-9

    Differential ripening-related demethylation of the promoter of RIN between pericarp and locular tissue. a RIN self-binding sites and differentially methylated regions in the RIN promoter, based on analyses of pericarp samples (available at ted.bti.cornell.edu/epigenome ). ChIP-seq plot depicts position of RIN binding, with peak height representing the number of reads covering each site (i.e., read depth). Red bars indicate cytosine methylation ratios at the equivalent position. Two promoter fragments analyzed by McrBC-PCR are indicated by black bars. b McrBC-PCR analysis of two RIN promoter fragments in pericarp (Pe) and locular tissue (Lo) at four stages spanning fruit expansion and ripening. A total of 0.5 μg genomic DNA was digested by McrBC (NEB) with GTP (+), or without GTP (−) as a negative control. An unmethylated region of the PME (Solyc03g123630) promoter is included as a positive control, and a highly methylated region of the CNR (Solyc02g077920) promoter showing complete digestion of methylated DNA. DPA, days post-anthesis; MG, mature green; Br, breaker; Pk, pink; RR, red ripe)
    Figure Legend Snippet: Differential ripening-related demethylation of the promoter of RIN between pericarp and locular tissue. a RIN self-binding sites and differentially methylated regions in the RIN promoter, based on analyses of pericarp samples (available at ted.bti.cornell.edu/epigenome ). ChIP-seq plot depicts position of RIN binding, with peak height representing the number of reads covering each site (i.e., read depth). Red bars indicate cytosine methylation ratios at the equivalent position. Two promoter fragments analyzed by McrBC-PCR are indicated by black bars. b McrBC-PCR analysis of two RIN promoter fragments in pericarp (Pe) and locular tissue (Lo) at four stages spanning fruit expansion and ripening. A total of 0.5 μg genomic DNA was digested by McrBC (NEB) with GTP (+), or without GTP (−) as a negative control. An unmethylated region of the PME (Solyc03g123630) promoter is included as a positive control, and a highly methylated region of the CNR (Solyc02g077920) promoter showing complete digestion of methylated DNA. DPA, days post-anthesis; MG, mature green; Br, breaker; Pk, pink; RR, red ripe)

    Techniques Used: Binding Assay, Methylation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Positive Control

    20) Product Images from "Independent functions of DNMT1 and USP7 at replication foci"

    Article Title: Independent functions of DNMT1 and USP7 at replication foci

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0179-z

    Ablation of USP7 does not affect steady-state level of DNMT1 or global DNA methylation. a MEFs that lack USP7 after Cre-mediated excision of a Floxed allele of Usp7 show normal levels of DNMT1 (lane 2) but no detectable USP7 protein. Biological replicates are shown as Experiments 1 and 2. b Normal expression of DNMT1 and UHRF1 in human H1299 lung carcinoma cells containing an inducible shRNA against USP7 mRNA. Ablation of USP7 has no detectable effect on expression of other proteins. c Normal steady-state expression of USP7 in the absence of DNMT1 in ES cells. d Normal DNA methylation in MEFs that lack USP7. McrBC cleaves methylated DNA; HpaII cleaves unmethylated DNA at CCGG sites; MspI cleaves CCGG sites whether methylated or unmethylated. DNA from wild-type and Usp7 − / − MEFs show similar patterns of resistance to both McrBC and HpaII. e Removal of USP7 by an inducible shRNA against USP7 from H1299 human lung carcinoma cells does not affect DNA methylation levels as assessed by resistance of DNA to HpaII. f LUMA analysis shows that DNA methylation is not measurably affected by removal of USP7. n = 2 (biological replicates). Error bars show standard deviations, center value is mean, and p values were calculated using the two-tailed t test
    Figure Legend Snippet: Ablation of USP7 does not affect steady-state level of DNMT1 or global DNA methylation. a MEFs that lack USP7 after Cre-mediated excision of a Floxed allele of Usp7 show normal levels of DNMT1 (lane 2) but no detectable USP7 protein. Biological replicates are shown as Experiments 1 and 2. b Normal expression of DNMT1 and UHRF1 in human H1299 lung carcinoma cells containing an inducible shRNA against USP7 mRNA. Ablation of USP7 has no detectable effect on expression of other proteins. c Normal steady-state expression of USP7 in the absence of DNMT1 in ES cells. d Normal DNA methylation in MEFs that lack USP7. McrBC cleaves methylated DNA; HpaII cleaves unmethylated DNA at CCGG sites; MspI cleaves CCGG sites whether methylated or unmethylated. DNA from wild-type and Usp7 − / − MEFs show similar patterns of resistance to both McrBC and HpaII. e Removal of USP7 by an inducible shRNA against USP7 from H1299 human lung carcinoma cells does not affect DNA methylation levels as assessed by resistance of DNA to HpaII. f LUMA analysis shows that DNA methylation is not measurably affected by removal of USP7. n = 2 (biological replicates). Error bars show standard deviations, center value is mean, and p values were calculated using the two-tailed t test

    Techniques Used: DNA Methylation Assay, Expressing, shRNA, Methylation, Two Tailed Test

    21) Product Images from "Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation"

    Article Title: Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp413

    Comparison of methylation analysis of cell lines. ( A–C ) shows the comparison of a normal fibroblast used to produce representations from two separate aliquots of DNA and the representations were analyzed and the intensity compared to each other by scatter plot; the intensities of each being on one and the other axis, and the correlation calculated: ( A ) being replicate hybs, (B) being technical replicates and ( C ) being biological replicates. ( D ) The comparison of the breast cancer cell line SKBR3 and the normal female fibroblast cell line as a scatter plot of the intensities from the SKBR3 experiment as the y -axis and the intensities of chp-skn-1 on the x -axis, and the correlation shown. ( E ) An example of bisulfite validation for a fragment found methylated in the tumor and not in the normal at a CpG island at chr 9:2197965–21980065, which lies upstream of the p14 gene. The original data showing the ratio differences is shown with the position of the fragment. To the right is the electropherogram identifying a region of cytosines that do not convert and at the bottom is a short table identifying the genomic position and number of McrBC site. ( F ) shows data for the cell line Huh7 compared to the normal cell line chp-skn-1 for the p16 gene CpG island graphed by genomic position. The x -axis is the genomic position of the probes and the y -axis is the ratio of two experiments, blue being chp-skn-1 and orange being Huh7. Inset above is shown is the relation of the transcript and inset below is the relation of the CpG island to the probes.
    Figure Legend Snippet: Comparison of methylation analysis of cell lines. ( A–C ) shows the comparison of a normal fibroblast used to produce representations from two separate aliquots of DNA and the representations were analyzed and the intensity compared to each other by scatter plot; the intensities of each being on one and the other axis, and the correlation calculated: ( A ) being replicate hybs, (B) being technical replicates and ( C ) being biological replicates. ( D ) The comparison of the breast cancer cell line SKBR3 and the normal female fibroblast cell line as a scatter plot of the intensities from the SKBR3 experiment as the y -axis and the intensities of chp-skn-1 on the x -axis, and the correlation shown. ( E ) An example of bisulfite validation for a fragment found methylated in the tumor and not in the normal at a CpG island at chr 9:2197965–21980065, which lies upstream of the p14 gene. The original data showing the ratio differences is shown with the position of the fragment. To the right is the electropherogram identifying a region of cytosines that do not convert and at the bottom is a short table identifying the genomic position and number of McrBC site. ( F ) shows data for the cell line Huh7 compared to the normal cell line chp-skn-1 for the p16 gene CpG island graphed by genomic position. The x -axis is the genomic position of the probes and the y -axis is the ratio of two experiments, blue being chp-skn-1 and orange being Huh7. Inset above is shown is the relation of the transcript and inset below is the relation of the CpG island to the probes.

    Techniques Used: Methylation

    Schematic of the procedure. Shown at the top is genomic DNA with a CpG island that is methylated. The DNA is cleaved with the restriction endonuclease MspI and adaptors ligated. The ligated material is divided evenly, one half being digested with McrBC and the other half being mock digested. This material is used as template for PCR amplification and the resulting product is used for microarray comparison.
    Figure Legend Snippet: Schematic of the procedure. Shown at the top is genomic DNA with a CpG island that is methylated. The DNA is cleaved with the restriction endonuclease MspI and adaptors ligated. The ligated material is divided evenly, one half being digested with McrBC and the other half being mock digested. This material is used as template for PCR amplification and the resulting product is used for microarray comparison.

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification, Microarray

    McrBC PCR of two different fragments of the MTSS1 CpG island for tumors identified of having methylation of this island compared to matched normal samples. Fragment 1 encompasses the MspI fragment we have identified as being methylated and overlaps the gene TSS. Fragment 5, we do not detect methylation. Both Normal and Tumor were digested with McrBC or mock digested for both matched pairs.
    Figure Legend Snippet: McrBC PCR of two different fragments of the MTSS1 CpG island for tumors identified of having methylation of this island compared to matched normal samples. Fragment 1 encompasses the MspI fragment we have identified as being methylated and overlaps the gene TSS. Fragment 5, we do not detect methylation. Both Normal and Tumor were digested with McrBC or mock digested for both matched pairs.

    Techniques Used: Polymerase Chain Reaction, Methylation

    22) Product Images from "Distinct Mechanisms Determine Transposon Inheritance and Methylation via Small Interfering RNA and Histone ModificationDistinct Mechanisms Control Transposon Inheritance through Overlapping Pathways"

    Article Title: Distinct Mechanisms Determine Transposon Inheritance and Methylation via Small Interfering RNA and Histone ModificationDistinct Mechanisms Control Transposon Inheritance through Overlapping Pathways

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0000067

    Inheritance of Transposon Modification Reverse-transcribed cDNA (A), ChIP (B), and McrBC-digested genomic DNA (C) were amplified by PCR using primers from five retroelements and one DNA transposon in mutant (m/m) and backcrossed plants (m/+). Primers corresponded to transcribed ORFs for each element except for AtMu1 ChIP, which was done on the terminal inverted repeat (TIR). For ATLANTYS2 , the larger product is ATLANTYS2-1 and smaller product is ATLANTYS2-2 . Input RNA was normalized for each genotype using actin primers. (A) Mock RT–PCR was performed without reverse transcriptase (−RT) using primers specific for the Cen180 repeat, which can detect trace amounts of contaminating DNA due to its high-copy number. (B) ChIP was performed with antibodies recognizing dimethyl lysine-9 (K9) and dimethyl lysine-4 (K4) of histone H3 along with no antibody (na) and total (T) DNA controls. ChIP analysis for AtMu1 and ATCOPIA4 was performed using reduced cycles of PCR and Southern blotting (see Materials and Methods ). (C) McrPCR was carried out on untreated (−) and McrBC-treated (+) DNA (see Materials and Methods ).
    Figure Legend Snippet: Inheritance of Transposon Modification Reverse-transcribed cDNA (A), ChIP (B), and McrBC-digested genomic DNA (C) were amplified by PCR using primers from five retroelements and one DNA transposon in mutant (m/m) and backcrossed plants (m/+). Primers corresponded to transcribed ORFs for each element except for AtMu1 ChIP, which was done on the terminal inverted repeat (TIR). For ATLANTYS2 , the larger product is ATLANTYS2-1 and smaller product is ATLANTYS2-2 . Input RNA was normalized for each genotype using actin primers. (A) Mock RT–PCR was performed without reverse transcriptase (−RT) using primers specific for the Cen180 repeat, which can detect trace amounts of contaminating DNA due to its high-copy number. (B) ChIP was performed with antibodies recognizing dimethyl lysine-9 (K9) and dimethyl lysine-4 (K4) of histone H3 along with no antibody (na) and total (T) DNA controls. ChIP analysis for AtMu1 and ATCOPIA4 was performed using reduced cycles of PCR and Southern blotting (see Materials and Methods ). (C) McrPCR was carried out on untreated (−) and McrBC-treated (+) DNA (see Materials and Methods ).

    Techniques Used: Modification, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Southern Blot

    23) Product Images from "Importance of parental genome balance in the generation of novel yet heritable epigenetic and transcriptional states during doubled haploid breeding"

    Article Title: Importance of parental genome balance in the generation of novel yet heritable epigenetic and transcriptional states during doubled haploid breeding

    Journal: bioRxiv

    doi: 10.1101/812347

    Intersection of DMRs and Genomic features in B. oleracea DHs. a) Venn diagram showing extent of DMR and gene overlap in base pairs. b) The three DMR contexts have different distributions across the genes and flanking regions with which they overlap. Density plot showing the distribution of dhDMRs in each sequence context across the genes they overlap with. c) Example of the most highly correlated expression and methylation for a gene, AGAMOUS-LIKE (Bo6g014360). Left side of the plot shows a single nucleotide resolution plot of methylation across the region, the right side of the plot shows gene expression. d) McrBC assay showing the targeted removal of DNA methylation at the Bo6g014360-DMR using dCas9-TET3CD. e) RT-PCR assay showing an enhancement in Bo6g014360 transcription upon removal of DNA methylation using dCas9-TET3CD.
    Figure Legend Snippet: Intersection of DMRs and Genomic features in B. oleracea DHs. a) Venn diagram showing extent of DMR and gene overlap in base pairs. b) The three DMR contexts have different distributions across the genes and flanking regions with which they overlap. Density plot showing the distribution of dhDMRs in each sequence context across the genes they overlap with. c) Example of the most highly correlated expression and methylation for a gene, AGAMOUS-LIKE (Bo6g014360). Left side of the plot shows a single nucleotide resolution plot of methylation across the region, the right side of the plot shows gene expression. d) McrBC assay showing the targeted removal of DNA methylation at the Bo6g014360-DMR using dCas9-TET3CD. e) RT-PCR assay showing an enhancement in Bo6g014360 transcription upon removal of DNA methylation using dCas9-TET3CD.

    Techniques Used: Sequencing, Expressing, Methylation, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction

    24) Product Images from "Novel features of telomere biology revealed by the absence of telomeric DNA methylation"

    Article Title: Novel features of telomere biology revealed by the absence of telomeric DNA methylation

    Journal: Genome Research

    doi: 10.1101/gr.202465.115

    Methylation-dependent restriction enzyme analyses confirm the absence of telomeric DNA methylation. ( A ) Cartoon representing the sequence specificity of the restriction enzymes used to digest Arabidopsis genomic DNA. ( B ) Southern blot hybridizations of Arabidopsis genomic DNA digested with the restriction enzymes indicated in A . The upper panel shows equal amounts of undigested (Control) or digested (HpaII, FspEI, or McrBC) DNA samples hybridized with a telomeric probe. The four DNA samples were also digested with Tru9I prior to hybridization. The middle panel shows the hybridization of the same samples with a 180-bp centromeric repeat probe. The ethidium bromide staining of the samples is shown in the lower panel. The control DNA sample was run in the same gel as the rest of the samples, so that their corresponding hybridization signals were processed equally. ( C ) Bar plot representation of the telomeric and centromeric bottom band hybridization signals expressed as percentages of the control. C, H, F, and M represent Control, HpaII, FspEI, and McrBC, respectively.
    Figure Legend Snippet: Methylation-dependent restriction enzyme analyses confirm the absence of telomeric DNA methylation. ( A ) Cartoon representing the sequence specificity of the restriction enzymes used to digest Arabidopsis genomic DNA. ( B ) Southern blot hybridizations of Arabidopsis genomic DNA digested with the restriction enzymes indicated in A . The upper panel shows equal amounts of undigested (Control) or digested (HpaII, FspEI, or McrBC) DNA samples hybridized with a telomeric probe. The four DNA samples were also digested with Tru9I prior to hybridization. The middle panel shows the hybridization of the same samples with a 180-bp centromeric repeat probe. The ethidium bromide staining of the samples is shown in the lower panel. The control DNA sample was run in the same gel as the rest of the samples, so that their corresponding hybridization signals were processed equally. ( C ) Bar plot representation of the telomeric and centromeric bottom band hybridization signals expressed as percentages of the control. C, H, F, and M represent Control, HpaII, FspEI, and McrBC, respectively.

    Techniques Used: Methylation, DNA Methylation Assay, Sequencing, Southern Blot, Hybridization, Staining

    25) Product Images from "CpG Methylation of Human Papillomavirus Type 16 DNA in Cervical Cancer Cell Lines and in Clinical Specimens: Genomic Hypomethylation Correlates with Carcinogenic Progression"

    Article Title: CpG Methylation of Human Papillomavirus Type 16 DNA in Cervical Cancer Cell Lines and in Clinical Specimens: Genomic Hypomethylation Correlates with Carcinogenic Progression

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.11.6227-6234.2003

    McrBC cleavage of segments of the HPV-16 genomes in SiHa and CaSki cells and three clinical samples detects patterns of hyper- and hypomethylation. (A) Genomic map of HPV-16 (7,906 bp), with genes E6, E7, E1, E2, E5, L2, and L1, the LCR, and the relative locations of amplicons G1 to G8 indicated. (B) Cleavage of amplicons G1 to G8 by McrBC (right lane of each pair of samples) indicates methylation, compared with uncleaved controls (left lane of each pair). The cleavage pattern indicates hypermethylation throughout most HPV-16 genomes in DNA from CaSki cells and tumor 6, hypermethylation of the late genes in the single HPV-16 genome of SiHa cells, and hypo- or no methylation in tumor 4 and a CIN I lesion.
    Figure Legend Snippet: McrBC cleavage of segments of the HPV-16 genomes in SiHa and CaSki cells and three clinical samples detects patterns of hyper- and hypomethylation. (A) Genomic map of HPV-16 (7,906 bp), with genes E6, E7, E1, E2, E5, L2, and L1, the LCR, and the relative locations of amplicons G1 to G8 indicated. (B) Cleavage of amplicons G1 to G8 by McrBC (right lane of each pair of samples) indicates methylation, compared with uncleaved controls (left lane of each pair). The cleavage pattern indicates hypermethylation throughout most HPV-16 genomes in DNA from CaSki cells and tumor 6, hypermethylation of the late genes in the single HPV-16 genome of SiHa cells, and hypo- or no methylation in tumor 4 and a CIN I lesion.

    Techniques Used: Methylation

    26) Product Images from "An Epigenetic LTR-retrotransposon insertion in the upstream region of BnSHP1.A9 controls quantitative pod shattering resistance in Brassica napus"

    Article Title: An Epigenetic LTR-retrotransposon insertion in the upstream region of BnSHP1.A9 controls quantitative pod shattering resistance in Brassica napus

    Journal: bioRxiv

    doi: 10.1101/858407

    Cross validation of DNA methylation status of BnSHP1.A9 in R1/R2 silique genomic DNA. A, amplified fragments illustrated in BnSHP1.A9 DNA from R1/R2 B, D : chop-PCR with BnSHP1.A9 gDNA template and McrBC digested gDNA of 20 DAF silique from R1 without McrBC digestion; a1: −2412/−1542, a2: −1616/−882, a3: −902/−30, a4: −49/+1413, a5: +1034/+2007 C, E : chop-PCR with BnSHP1.A9 gDNA template and McrBC digested gDNA of 20 DAF silique from R2. b1: −7412/−6823, b2: −6753/−5765, b3: −3191/−2285, b4: −2538/−1788, b5: −1707/−1027, b6: −1035/−102, b7: −49/+436, b8: +1035/+2012, b9: +1883/+2793 M: Trans 2k plus.
    Figure Legend Snippet: Cross validation of DNA methylation status of BnSHP1.A9 in R1/R2 silique genomic DNA. A, amplified fragments illustrated in BnSHP1.A9 DNA from R1/R2 B, D : chop-PCR with BnSHP1.A9 gDNA template and McrBC digested gDNA of 20 DAF silique from R1 without McrBC digestion; a1: −2412/−1542, a2: −1616/−882, a3: −902/−30, a4: −49/+1413, a5: +1034/+2007 C, E : chop-PCR with BnSHP1.A9 gDNA template and McrBC digested gDNA of 20 DAF silique from R2. b1: −7412/−6823, b2: −6753/−5765, b3: −3191/−2285, b4: −2538/−1788, b5: −1707/−1027, b6: −1035/−102, b7: −49/+436, b8: +1035/+2012, b9: +1883/+2793 M: Trans 2k plus.

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

    27) Product Images from "A Comprehensive Analysis of Allelic Methylation Status of CpG Islands on Human Chromosome 21q"

    Article Title: A Comprehensive Analysis of Allelic Methylation Status of CpG Islands on Human Chromosome 21q

    Journal: Genome Research

    doi: 10.1101/gr.1351604

    Mosaicism in maternal allele-specific methylation. ( A ) Maternal allele-specific methylation of CGI #59. Direct sequencing was performed using the PCR products from mock-treated ( bottom left ), HhaI- ( bottom center ), and McrBC-digested ( bottom right ) DNA isolated from PBLs. The arrowhead indicatesthe A/C SNP site. Note that the maternally inherited A allele wasdetected from both Hha I-treated (or methylated) DNA and McrBC-treated (or unmethylated) DNA. ( B ) Bisulfite genomic sequencing of CGI #59. Each row of circles corresponds to each clone of bisulfite PCR products. Open and closed circles stand for unmethylated and methylated C residues, respectively. The A/C SNP site is also indicated. Note that the clonesfor the maternally inherited A allele are composed of two populations, one completely methylated and the other completely escaping methylation.
    Figure Legend Snippet: Mosaicism in maternal allele-specific methylation. ( A ) Maternal allele-specific methylation of CGI #59. Direct sequencing was performed using the PCR products from mock-treated ( bottom left ), HhaI- ( bottom center ), and McrBC-digested ( bottom right ) DNA isolated from PBLs. The arrowhead indicatesthe A/C SNP site. Note that the maternally inherited A allele wasdetected from both Hha I-treated (or methylated) DNA and McrBC-treated (or unmethylated) DNA. ( B ) Bisulfite genomic sequencing of CGI #59. Each row of circles corresponds to each clone of bisulfite PCR products. Open and closed circles stand for unmethylated and methylated C residues, respectively. The A/C SNP site is also indicated. Note that the clonesfor the maternally inherited A allele are composed of two populations, one completely methylated and the other completely escaping methylation.

    Techniques Used: Methylation, Sequencing, Polymerase Chain Reaction, Isolation, Genomic Sequencing

    Allele-specific, parental-origin-independent methylation of CGI #130. Direct sequencing was performed using the PCR products from mock-treated ( bottom left ), HhaI- ( bottom center ), and McrBC-digested ( bottom right ) DNA. In the pedigree shown in A , the paternally inherited C allele ismethylated. On the other hand, the maternally inherited C allele ismethylated in the pedigree shown in B .
    Figure Legend Snippet: Allele-specific, parental-origin-independent methylation of CGI #130. Direct sequencing was performed using the PCR products from mock-treated ( bottom left ), HhaI- ( bottom center ), and McrBC-digested ( bottom right ) DNA. In the pedigree shown in A , the paternally inherited C allele ismethylated. On the other hand, the maternally inherited C allele ismethylated in the pedigree shown in B .

    Techniques Used: Methylation, Sequencing, Polymerase Chain Reaction

    Maternal allele-specific methylation pinpointed to tandem repeats. ( A ) Maternal allele-specific methylation of CGI #112. A map of CGI #112 (500 bp) isshown on the top with the positionsof A/T and C/T SNPs(i.e., SNP1 and SNP2). The arrowsin the map indicate the tandem repeats. The CGI wasPCR-amplified from untreated ( bottom left ), HhaI-digested ( bottom center ), and McrBC-digested ( bottom right ) genomic DNA isolated from PBLs of a C/T heterozygote at SNP2. The amplified productswere subjected to direct sequencing. The vertical arrowhead in each electropherogram denotes the SNP2 (C/T) sites. ( B ) Bisulfite genomic sequencing of CGI #112. Each row of circles corresponds to each clone of bisulfite PCR products. Open and closed circles stand for unmethylated and methylated C residues, respectively. The SNP1 (A/T) site is indicated by the arrowhead.
    Figure Legend Snippet: Maternal allele-specific methylation pinpointed to tandem repeats. ( A ) Maternal allele-specific methylation of CGI #112. A map of CGI #112 (500 bp) isshown on the top with the positionsof A/T and C/T SNPs(i.e., SNP1 and SNP2). The arrowsin the map indicate the tandem repeats. The CGI wasPCR-amplified from untreated ( bottom left ), HhaI-digested ( bottom center ), and McrBC-digested ( bottom right ) genomic DNA isolated from PBLs of a C/T heterozygote at SNP2. The amplified productswere subjected to direct sequencing. The vertical arrowhead in each electropherogram denotes the SNP2 (C/T) sites. ( B ) Bisulfite genomic sequencing of CGI #112. Each row of circles corresponds to each clone of bisulfite PCR products. Open and closed circles stand for unmethylated and methylated C residues, respectively. The SNP1 (A/T) site is indicated by the arrowhead.

    Techniques Used: Methylation, Amplification, Isolation, Sequencing, Genomic Sequencing, Polymerase Chain Reaction

    28) Product Images from "Light signaling controls nuclear architecture reorganization during seedling establishment"

    Article Title: Light signaling controls nuclear architecture reorganization during seedling establishment

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

    doi: 10.1073/pnas.1503512112

    Activity of the TGS pathway during photomorphogenesis. ( A ) RT-qPCR analysis of the indicated transcripts in cotyledons of 5-d-old seedlings grown in dark or light conditions. RNA levels are shown relative to the wild-type Light sample. Error bars correspond to the SEM from two biological replicates. ( B ) Cytosine methylation levels on AtGP1 ). The data are representative of two independent biological replicates. For each analysis, n > 30. ( C ) Effect of zebularine on the LTR AtGP1 ::GUS transcriptional reporter. GUS activity was tested in light- and dark-grown seedlings at 5 dpi grown with (+) or without (−) 50 μM of zebularine. (Scale bars, 0.5 mm.) ( D ) Cytosine methylation levels on 180-bp centromeric repeats performed as in B . ( E ) McrBC-PCR analysis of methylation levels on CACTA transposons and centromeric 180-bp repeats. ACTIN2 is used as a control for nonmethylated DNA. ( F ) DNA methylation analysis of the indicated TEs by McrBC-qPCR. At4g04020 gene TE border is given as controls for nonmethylated DNA, whereas FWA TE corresponds to heavily methylated repeats located upstream of the FWA gene. Error bars represent the SEM from two biological replicates.
    Figure Legend Snippet: Activity of the TGS pathway during photomorphogenesis. ( A ) RT-qPCR analysis of the indicated transcripts in cotyledons of 5-d-old seedlings grown in dark or light conditions. RNA levels are shown relative to the wild-type Light sample. Error bars correspond to the SEM from two biological replicates. ( B ) Cytosine methylation levels on AtGP1 ). The data are representative of two independent biological replicates. For each analysis, n > 30. ( C ) Effect of zebularine on the LTR AtGP1 ::GUS transcriptional reporter. GUS activity was tested in light- and dark-grown seedlings at 5 dpi grown with (+) or without (−) 50 μM of zebularine. (Scale bars, 0.5 mm.) ( D ) Cytosine methylation levels on 180-bp centromeric repeats performed as in B . ( E ) McrBC-PCR analysis of methylation levels on CACTA transposons and centromeric 180-bp repeats. ACTIN2 is used as a control for nonmethylated DNA. ( F ) DNA methylation analysis of the indicated TEs by McrBC-qPCR. At4g04020 gene TE border is given as controls for nonmethylated DNA, whereas FWA TE corresponds to heavily methylated repeats located upstream of the FWA gene. Error bars represent the SEM from two biological replicates.

    Techniques Used: Activity Assay, Quantitative RT-PCR, Methylation, Polymerase Chain Reaction, DNA Methylation Assay, Real-time Polymerase Chain Reaction

    29) Product Images from "Global increase in DNA methylation during orange fruit development and ripening"

    Article Title: Global increase in DNA methylation during orange fruit development and ripening

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

    doi: 10.1073/pnas.1815441116

    The significance of DNA methylation for orange fruit ripening. ( A ) Pictures of sweet oranges that were treated with DNA methylation inhibitor, 5-azacytidine (5′-Aza), or mock (ddH 2 O). The treated areas are circled. ( B ) DNA methylation levels of promoter regions of Cs3g20300 and Cs4g09560. McrBC-qPCR analysis was performed in Cs1, Cs5, and mock and 5′-Aza–treated samples. Methylated DNA can be digested by McrBC, thus higher qPCR signals indicate lower methylation levels. ( C ) Gene expression of Cs3g20300 and Cs4g09560 in untreated Cs1 and Cs5 fruits, and in mock and 5′-Aza treated fruits. * P
    Figure Legend Snippet: The significance of DNA methylation for orange fruit ripening. ( A ) Pictures of sweet oranges that were treated with DNA methylation inhibitor, 5-azacytidine (5′-Aza), or mock (ddH 2 O). The treated areas are circled. ( B ) DNA methylation levels of promoter regions of Cs3g20300 and Cs4g09560. McrBC-qPCR analysis was performed in Cs1, Cs5, and mock and 5′-Aza–treated samples. Methylated DNA can be digested by McrBC, thus higher qPCR signals indicate lower methylation levels. ( C ) Gene expression of Cs3g20300 and Cs4g09560 in untreated Cs1 and Cs5 fruits, and in mock and 5′-Aza treated fruits. * P

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

    30) Product Images from "Epigenetic Natural Variation in Arabidopsis thaliana"

    Article Title: Epigenetic Natural Variation in Arabidopsis thaliana

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0050174

    Heritability of Polymorphic Gene Methylation The gene At4g28850 is methylated in Col but not Ler. Genomic DNA was prepared from F 1 and F 2 siblings derived from reciprocal crosses between Col and Ler and subjected to McrBC digestion and PCR amplification of this locus as in Figure 1 . The amplification product (tb63b02) has a small deletion in Ler, enabling the parental alleles to be distinguished. DNA samples were digested (+) or mock-digested (−) with McrBC. Control primers were used to amplify a methylated retrotransposon (TA2) in each sample, as well as an unmethylated control tile (ta25c11). Samples of two F 1 and ten F 2 plants are shown for each cross (out of a total of eight and 40, respectively) ( Table S3 ). In almost all cases, the Col allele is digested by McrBC, and the Ler allele is never digested. Two exceptions are indicated (a and b) in which the Col allele has lost all or most of its associated methylation.
    Figure Legend Snippet: Heritability of Polymorphic Gene Methylation The gene At4g28850 is methylated in Col but not Ler. Genomic DNA was prepared from F 1 and F 2 siblings derived from reciprocal crosses between Col and Ler and subjected to McrBC digestion and PCR amplification of this locus as in Figure 1 . The amplification product (tb63b02) has a small deletion in Ler, enabling the parental alleles to be distinguished. DNA samples were digested (+) or mock-digested (−) with McrBC. Control primers were used to amplify a methylated retrotransposon (TA2) in each sample, as well as an unmethylated control tile (ta25c11). Samples of two F 1 and ten F 2 plants are shown for each cross (out of a total of eight and 40, respectively) ( Table S3 ). In almost all cases, the Col allele is digested by McrBC, and the Ler allele is never digested. Two exceptions are indicated (a and b) in which the Col allele has lost all or most of its associated methylation.

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

    Methylation Profiles for Col and Ler Arabidopsis Ecotypes (A) Microarray data from Chromosome 4 are displayed for a 125-kb region 9 Mb from the nuclear organizing region ( Figure S1 ). Open reading frames from genes (yellow) and retrotransposons (green) are indicated, along with repeats predicted by RepBase and TandemRepeatFinder. Small RNA matches from massively parallel signature-sequencing (MPSS) data are indicated. Tiles that represent significant CNPs are highlighted in purple (CGH), while tiles that detect significant DNA methylation are highlighted in red for the two ecotypes (5 mC, Col and Ler). Examples of a gene CNP (a), a TE CNP (b), and a methylation polymorphism (c) are boxed. (B) Significant methylation was detected by microarray analysis for two representative genes in Ler (At4g40980) and Col (At4g28850), respectively. This methylation was verified by digestion of genomic DNA by McrBC, followed by PCR amplification using primers specific for each gene (lower panels). Failure to amplify a product after digestion by McrBC indicates that the gene is methylated. Control primers from an unmethylated tile (ta25c11) and a methylated transposon (TA2) indicate complete digestion and amplification in each case.
    Figure Legend Snippet: Methylation Profiles for Col and Ler Arabidopsis Ecotypes (A) Microarray data from Chromosome 4 are displayed for a 125-kb region 9 Mb from the nuclear organizing region ( Figure S1 ). Open reading frames from genes (yellow) and retrotransposons (green) are indicated, along with repeats predicted by RepBase and TandemRepeatFinder. Small RNA matches from massively parallel signature-sequencing (MPSS) data are indicated. Tiles that represent significant CNPs are highlighted in purple (CGH), while tiles that detect significant DNA methylation are highlighted in red for the two ecotypes (5 mC, Col and Ler). Examples of a gene CNP (a), a TE CNP (b), and a methylation polymorphism (c) are boxed. (B) Significant methylation was detected by microarray analysis for two representative genes in Ler (At4g40980) and Col (At4g28850), respectively. This methylation was verified by digestion of genomic DNA by McrBC, followed by PCR amplification using primers specific for each gene (lower panels). Failure to amplify a product after digestion by McrBC indicates that the gene is methylated. Control primers from an unmethylated tile (ta25c11) and a methylated transposon (TA2) indicate complete digestion and amplification in each case.

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

    Methylation is Localized within Genes (A) Intermediate levels of methylation were detected by microarray analysis in Col and Ler for one representative gene (genome browser tracks are annotated as in Figure 1 ). Primer pairs 1–6 are indicated below each tile of the array. Amplification of McrBC digested Col DNA (+) and undigested Col DNA (−) was performed as in Figure 1 . Failure to amplify digested DNA indicated that methylation was localized to regions 2, 4, and 6, rather than spanning the entire gene. (B) The number of genic tiles detecting significant methylation was calculated at 10% intervals relative to the length of each gene and compared with the number expected if methylation was randomly distributed (black line). Only genes larger than 2 kb (three tiles or more per gene) were considered. Methylated tiles differing between ecotypes were also plotted in a similar way (light gray line). Genic methylation is largely concentrated in the middle of genes. For comparison, methylation distribution as a function of position was also calculated for TE-derived open reading frames (dark grey line) and is uniformly distributed.
    Figure Legend Snippet: Methylation is Localized within Genes (A) Intermediate levels of methylation were detected by microarray analysis in Col and Ler for one representative gene (genome browser tracks are annotated as in Figure 1 ). Primer pairs 1–6 are indicated below each tile of the array. Amplification of McrBC digested Col DNA (+) and undigested Col DNA (−) was performed as in Figure 1 . Failure to amplify digested DNA indicated that methylation was localized to regions 2, 4, and 6, rather than spanning the entire gene. (B) The number of genic tiles detecting significant methylation was calculated at 10% intervals relative to the length of each gene and compared with the number expected if methylation was randomly distributed (black line). Only genes larger than 2 kb (three tiles or more per gene) were considered. Methylated tiles differing between ecotypes were also plotted in a similar way (light gray line). Genic methylation is largely concentrated in the middle of genes. For comparison, methylation distribution as a function of position was also calculated for TE-derived open reading frames (dark grey line) and is uniformly distributed.

    Techniques Used: Methylation, Microarray, Amplification, Derivative Assay

    31) Product Images from "Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci"

    Article Title: Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.116.038687

    Methylation-dependent restriction digest performed on genomic PLAGL1 DNA prior to PCR. Genotyping result obtained from PCR amplification of DNA sample NA20588 after treatment with Hpa II (A) or McrBC (B). Genotyping result obtained from PCR amplification of DNA sample NA19312 after treatment with Hpa II (C) or McrBC (D).
    Figure Legend Snippet: Methylation-dependent restriction digest performed on genomic PLAGL1 DNA prior to PCR. Genotyping result obtained from PCR amplification of DNA sample NA20588 after treatment with Hpa II (A) or McrBC (B). Genotyping result obtained from PCR amplification of DNA sample NA19312 after treatment with Hpa II (C) or McrBC (D).

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification

    32) Product Images from "Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation"

    Article Title: Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation

    Journal: Developmental Biology

    doi: 10.1016/j.ydbio.2013.09.020

    Lack of evidence for a role for DNA methylation in pASC biology Smed-dnmt- 2 is expressed in the germ line and germ line stem cells (A) Scale bars, 500 µm. gDNA digestion with the methylation dependent restriction enzyme McrBC does not indicate any DNA cytosine methylation in the Schmidtea mediterranea genome as compared with that of humans. −: no enzyme; +: with enzyme (B). Control chromatogram after HPLC-MS displaying the sensitivity of nucleosides detection, in particular for 5 m C. (C) A representative chromatogram of S. mediterranea nucleoside analysis indicating the lack of 5 m C (D).
    Figure Legend Snippet: Lack of evidence for a role for DNA methylation in pASC biology Smed-dnmt- 2 is expressed in the germ line and germ line stem cells (A) Scale bars, 500 µm. gDNA digestion with the methylation dependent restriction enzyme McrBC does not indicate any DNA cytosine methylation in the Schmidtea mediterranea genome as compared with that of humans. −: no enzyme; +: with enzyme (B). Control chromatogram after HPLC-MS displaying the sensitivity of nucleosides detection, in particular for 5 m C. (C) A representative chromatogram of S. mediterranea nucleoside analysis indicating the lack of 5 m C (D).

    Techniques Used: DNA Methylation Assay, Methylation, High Performance Liquid Chromatography, Mass Spectrometry

    33) Product Images from "Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana"

    Article Title: Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana

    Journal: Silence

    doi: 10.1186/1758-907X-3-6

    Molecular characteristics of LUCH associated with RdDM. (A) Analysis of DNA methylation in the d35S and the LUC coding region in LUCH by McrBC-PCR. The two d35S fragments are as diagrammed in Figure 1 . − and + indicate McrBC-untreated and treated genomic DNA, respectively. ‘H 2 O’ is a negative control PCR without genomic DNA. McrBC digests methylated DNA to result in reduced PCR product amounts. (B) Bisulfite sequencing analysis of cytosine methylation in d35S in LUCH in wild type and ago4–6 . The top strand of d35S #3 in Figure 1 was analyzed. (C) d35S -specific siRNA accumulation in the LUCH line as detected by northern blotting. The numbers indicate the amount of enriched small RNAs loaded into the gel. Col-0, wild type (with no transgene).
    Figure Legend Snippet: Molecular characteristics of LUCH associated with RdDM. (A) Analysis of DNA methylation in the d35S and the LUC coding region in LUCH by McrBC-PCR. The two d35S fragments are as diagrammed in Figure 1 . − and + indicate McrBC-untreated and treated genomic DNA, respectively. ‘H 2 O’ is a negative control PCR without genomic DNA. McrBC digests methylated DNA to result in reduced PCR product amounts. (B) Bisulfite sequencing analysis of cytosine methylation in d35S in LUCH in wild type and ago4–6 . The top strand of d35S #3 in Figure 1 was analyzed. (C) d35S -specific siRNA accumulation in the LUCH line as detected by northern blotting. The numbers indicate the amount of enriched small RNAs loaded into the gel. Col-0, wild type (with no transgene).

    Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Negative Control, Methylation, Methylation Sequencing, Northern Blot

    Structure of LUCH and its neighboring transgene. RB and LB, right border and left border of the T-DNA, respectively. The arrows indicate the directions of the coding regions. The d35S fragments (marked #1 to #3) specific for the d35S promoter upstream of LUC are amplified by PCR following digestion with the restriction enzyme McrBC as well as in bisulfite sequencing.
    Figure Legend Snippet: Structure of LUCH and its neighboring transgene. RB and LB, right border and left border of the T-DNA, respectively. The arrows indicate the directions of the coding regions. The d35S fragments (marked #1 to #3) specific for the d35S promoter upstream of LUC are amplified by PCR following digestion with the restriction enzyme McrBC as well as in bisulfite sequencing.

    Techniques Used: Amplification, Polymerase Chain Reaction, Methylation Sequencing

    34) Product Images from "Identification of Novel High-Frequency DNA Methylation Changes in Breast Cancer"

    Article Title: Identification of Novel High-Frequency DNA Methylation Changes in Breast Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001314

    Differential DNA methylation of identified loci within an initial validation panel of clinical samples. qPCR measurements of DNA methylation density were obtained for 53 loci across 16 IDC tumor samples and 25 normal or benign breast samples. An average delta Ct (Ct McrBC –Ct Mock ) less than 1.0 was scored as sparsely methylated (green cells). A delta Ct of 1.0 indicates that approximately half of the DNA in the reaction was cleaved by McrBC within the amplified region and therefore contained a measurable density of DNA methylation. An average delta Ct greater than or equal to 1.0, but less than 2.0 was scored as intermediately methylated (yellow cells). Finally, an average delta Ct≥2.0 (≥75% of DNA molecules were cleaved by McrBC) was scored as densely methylated (red cells).
    Figure Legend Snippet: Differential DNA methylation of identified loci within an initial validation panel of clinical samples. qPCR measurements of DNA methylation density were obtained for 53 loci across 16 IDC tumor samples and 25 normal or benign breast samples. An average delta Ct (Ct McrBC –Ct Mock ) less than 1.0 was scored as sparsely methylated (green cells). A delta Ct of 1.0 indicates that approximately half of the DNA in the reaction was cleaved by McrBC within the amplified region and therefore contained a measurable density of DNA methylation. An average delta Ct greater than or equal to 1.0, but less than 2.0 was scored as intermediately methylated (yellow cells). Finally, an average delta Ct≥2.0 (≥75% of DNA molecules were cleaved by McrBC) was scored as densely methylated (red cells).

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

    Differential DNA methylation relative to tumor stage. (A) Comparison of the frequency of differential DNA methylation of 16 loci in stage I breast tumors relative to stage II–III breast tumors. (B) DNA methylation density of 3 selected loci relative to tumor stage. The percent depletion by McrBC for each sample in which a given locus scored as methylated was calculated [1-(1/2̂delta Ct (McrBC digested – Mock treated)) * 100] to provide a measure of the load of methylated molecules within the sample. The % depletion is plotted (from left to right) for normal and benign samples, stage I tumors, stage IIA tumors, stage IIB tumors and stage III tumors.
    Figure Legend Snippet: Differential DNA methylation relative to tumor stage. (A) Comparison of the frequency of differential DNA methylation of 16 loci in stage I breast tumors relative to stage II–III breast tumors. (B) DNA methylation density of 3 selected loci relative to tumor stage. The percent depletion by McrBC for each sample in which a given locus scored as methylated was calculated [1-(1/2̂delta Ct (McrBC digested – Mock treated)) * 100] to provide a measure of the load of methylated molecules within the sample. The % depletion is plotted (from left to right) for normal and benign samples, stage I tumors, stage IIA tumors, stage IIB tumors and stage III tumors.

    Techniques Used: DNA Methylation Assay, Methylation

    Related Articles

    Real-time Polymerase Chain Reaction:

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    Article Snippet: .. Validation of CpG methylation CpG methylation levels were examined by using Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) with McrBC restriction endonuclease (NEB, cat. no. M0272). ..

    Article Title: Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs
    Article Snippet: .. About 500 ng of DNA at 20 ng/μl was incubated with 20 U McrBC (New England Biolabs) or an equivalent volume of 50% glycerol for 2 h at 37°C followed by heat inactivation at 80°C for 20 min. Target regions were amplified by qPCR from 40-ng digested DNA using SYBR Green JumpStart Taq ReadyMix (Sigma S9194), using primers described in . .. Total DNA (500 ng) from parents and hybrids was treated with sodium bisulphite using EZ-DNA methylation gold kit (Zymo Research) as per manufacturer's instructions.

    Negative Control:

    Article Title: PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit
    Article Snippet: .. The isolated DNA was then digested with 40 units of the methylation-sensitive restriction enzyme McrBC (New England Biolabs; M0272L) for 2 h, and the digestion buffer without GTPase was used as the negative control. .. The methylation level in the digested DNA templates was measured by semiquantitative PCR.

    Article Title: High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening
    Article Snippet: .. A total of 0.5 μg DNA was digested with 7.5 U McrBC (NEB) according to the manufacturer’s recommendations for 6 h at 37 °C, or the same reaction without GTP as a negative control. .. PCR was performed using 40 ng McrBC-treated DNA.

    Amplification:

    Article Title: Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs
    Article Snippet: .. About 500 ng of DNA at 20 ng/μl was incubated with 20 U McrBC (New England Biolabs) or an equivalent volume of 50% glycerol for 2 h at 37°C followed by heat inactivation at 80°C for 20 min. Target regions were amplified by qPCR from 40-ng digested DNA using SYBR Green JumpStart Taq ReadyMix (Sigma S9194), using primers described in . .. Total DNA (500 ng) from parents and hybrids was treated with sodium bisulphite using EZ-DNA methylation gold kit (Zymo Research) as per manufacturer's instructions.

    Methylation:

    Article Title: Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential
    Article Snippet: .. Validation of CpG methylation CpG methylation levels were examined by using Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) with McrBC restriction endonuclease (NEB, cat. no. M0272). ..

    Article Title: PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit
    Article Snippet: .. The isolated DNA was then digested with 40 units of the methylation-sensitive restriction enzyme McrBC (New England Biolabs; M0272L) for 2 h, and the digestion buffer without GTPase was used as the negative control. .. The methylation level in the digested DNA templates was measured by semiquantitative PCR.

    Article Title: Independent functions of DNMT1 and USP7 at replication foci
    Article Snippet: .. Genomic DNA was digested for two rounds with methylation-sensitive enzyme HpaII, its isoschizomer MspI as a control, or McrBC (all from NEB). .. DNA was quantified and ran on 0.8% agarose gel, which was stained with ethidium bromide.

    Article Title: Chromatin and sequence features that define the fine and gross structure of genomic methylation patterns
    Article Snippet: .. Unmethylated and methylated compartments were obtained by limit digestions of 10–15 μg of DNA with McrBC or five tetranucleotide methylation-sensitive restriction endonucleases (referred to as RE), respectively (New England BioLabs). ..

    Isolation:

    Article Title: PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit
    Article Snippet: .. The isolated DNA was then digested with 40 units of the methylation-sensitive restriction enzyme McrBC (New England Biolabs; M0272L) for 2 h, and the digestion buffer without GTPase was used as the negative control. .. The methylation level in the digested DNA templates was measured by semiquantitative PCR.

    SYBR Green Assay:

    Article Title: Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs
    Article Snippet: .. About 500 ng of DNA at 20 ng/μl was incubated with 20 U McrBC (New England Biolabs) or an equivalent volume of 50% glycerol for 2 h at 37°C followed by heat inactivation at 80°C for 20 min. Target regions were amplified by qPCR from 40-ng digested DNA using SYBR Green JumpStart Taq ReadyMix (Sigma S9194), using primers described in . .. Total DNA (500 ng) from parents and hybrids was treated with sodium bisulphite using EZ-DNA methylation gold kit (Zymo Research) as per manufacturer's instructions.

    Incubation:

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    Article Title: Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs
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    CpG Methylation Assay:

    Article Title: Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential
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    The suvh1–1 mutation does not affect <t>DNA</t> methylation. ( A and B ) <t>McrBC-qPCR</t> analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.
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    The suvh1–1 mutation does not affect DNA methylation. ( A and B ) McrBC-qPCR analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.

    Journal: Nucleic Acids Research

    Article Title: SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation

    doi: 10.1093/nar/gkv958

    Figure Lengend Snippet: The suvh1–1 mutation does not affect DNA methylation. ( A and B ) McrBC-qPCR analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.

    Article Snippet: One hundred nanogram DNA was treated with 2 units of McrBC (New England Biolabs, M0272S) at 37°C for 30 min, and a mix without McrBC was performed in parallel as the control.

    Techniques: Mutagenesis, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Amplification, Sequencing

    qPCR validation of microarray results for remarkable genes showing significant changes in DNA methylation and transcript levels between d1 and d7 in neonatal murine hearts. The left panels show DNA methylation and gene expression microarray results represented by red bars and black markers, respectively; DNA methylation is expressed as MeDIP enrichment and gene expression levels as normalized microarray signals. The black markers preceding that indicating the neonatal d1 represent gene expression levels in embryonic hearts E16, E18, E19, E20. The middle panels present CpG methylation estimated with Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) where the DNA methylation levels correspond to the amounts of DNA undigested by CpG methylation dependent McrBC enzyme (1-(McrBC/Input). The right panels demonstrate transcript levels determined with qPCR as the ratios to the reference transcript of the Tbp gene (TATA binding protein gene). The microarray results were determined for pooled samples of 3 mice. The qPCR results represent average values obtained for three individuals for each developmental time-point. The statistical significance has been determined with two-tailed heteroscedastic Student’s t -test. The complete results of qPCR validation are collected in Additional file 2 : F2

    Journal: BMC Genomics

    Article Title: Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential

    doi: 10.1186/s12864-016-2545-1

    Figure Lengend Snippet: qPCR validation of microarray results for remarkable genes showing significant changes in DNA methylation and transcript levels between d1 and d7 in neonatal murine hearts. The left panels show DNA methylation and gene expression microarray results represented by red bars and black markers, respectively; DNA methylation is expressed as MeDIP enrichment and gene expression levels as normalized microarray signals. The black markers preceding that indicating the neonatal d1 represent gene expression levels in embryonic hearts E16, E18, E19, E20. The middle panels present CpG methylation estimated with Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) where the DNA methylation levels correspond to the amounts of DNA undigested by CpG methylation dependent McrBC enzyme (1-(McrBC/Input). The right panels demonstrate transcript levels determined with qPCR as the ratios to the reference transcript of the Tbp gene (TATA binding protein gene). The microarray results were determined for pooled samples of 3 mice. The qPCR results represent average values obtained for three individuals for each developmental time-point. The statistical significance has been determined with two-tailed heteroscedastic Student’s t -test. The complete results of qPCR validation are collected in Additional file 2 : F2

    Article Snippet: Validation of CpG methylation CpG methylation levels were examined by using Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) with McrBC restriction endonuclease (NEB, cat. no. M0272).

    Techniques: Real-time Polymerase Chain Reaction, Microarray, DNA Methylation Assay, Expressing, Methylated DNA Immunoprecipitation, CpG Methylation Assay, Methylation, Binding Assay, Mouse Assay, Two Tailed Test

    Differential ripening-related demethylation of the promoter of RIN between pericarp and locular tissue. a RIN self-binding sites and differentially methylated regions in the RIN ). ChIP-seq plot depicts position of RIN binding, with peak height representing the number of reads covering each site (i.e., read depth). Red bars indicate cytosine methylation ratios at the equivalent position. Two promoter fragments analyzed by McrBC-PCR are indicated by black bars. b McrBC-PCR analysis of two RIN promoter fragments in pericarp (Pe) and locular tissue (Lo) at four stages spanning fruit expansion and ripening. A total of 0.5 μg genomic DNA was digested by McrBC (NEB) with GTP (+), or without GTP (−) as a negative control. An unmethylated region of the PME (Solyc03g123630) promoter is included as a positive control, and a highly methylated region of the CNR (Solyc02g077920) promoter showing complete digestion of methylated DNA. DPA, days post-anthesis; MG, mature green; Br, breaker; Pk, pink; RR, red ripe)

    Journal: Nature Communications

    Article Title: High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening

    doi: 10.1038/s41467-017-02782-9

    Figure Lengend Snippet: Differential ripening-related demethylation of the promoter of RIN between pericarp and locular tissue. a RIN self-binding sites and differentially methylated regions in the RIN ). ChIP-seq plot depicts position of RIN binding, with peak height representing the number of reads covering each site (i.e., read depth). Red bars indicate cytosine methylation ratios at the equivalent position. Two promoter fragments analyzed by McrBC-PCR are indicated by black bars. b McrBC-PCR analysis of two RIN promoter fragments in pericarp (Pe) and locular tissue (Lo) at four stages spanning fruit expansion and ripening. A total of 0.5 μg genomic DNA was digested by McrBC (NEB) with GTP (+), or without GTP (−) as a negative control. An unmethylated region of the PME (Solyc03g123630) promoter is included as a positive control, and a highly methylated region of the CNR (Solyc02g077920) promoter showing complete digestion of methylated DNA. DPA, days post-anthesis; MG, mature green; Br, breaker; Pk, pink; RR, red ripe)

    Article Snippet: A total of 0.5 μg DNA was digested with 7.5 U McrBC (NEB) according to the manufacturer’s recommendations for 6 h at 37 °C, or the same reaction without GTP as a negative control.

    Techniques: Binding Assay, Methylation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Positive Control

    Methylation levels of the PbGA2ox8 promoter regions among “Red Zaosu”, “Zaosu”, and their progeny as assessed by McrBc-PCR. The 1300-bp region upstream of the ATG translational start site of PbGA2ox8 was divided into four overlapping fragments: promoter region 1 ( pG28-1 ), promoter region 2 ( pG28-2 ), promoter region 3 ( pG28-3 ), and promoter region 4 ( pG28-4 ). In total, 0.5 μg of genomic DNA was digested with McrBc in the presence GTP (+). Samples lacking GTP served as the negative controls (−). F1R and F1G, pooled red-, and green-leafed segregants, respectively, from the 15F1 population; F1R1–11 and F1G1–11, 11 randomly selected red and green-leafed individuals, respectively, from the 15F1 population; +1, ATG translational start site.

    Journal: Horticulture Research

    Article Title: PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit

    doi: 10.1038/s41438-019-0220-9

    Figure Lengend Snippet: Methylation levels of the PbGA2ox8 promoter regions among “Red Zaosu”, “Zaosu”, and their progeny as assessed by McrBc-PCR. The 1300-bp region upstream of the ATG translational start site of PbGA2ox8 was divided into four overlapping fragments: promoter region 1 ( pG28-1 ), promoter region 2 ( pG28-2 ), promoter region 3 ( pG28-3 ), and promoter region 4 ( pG28-4 ). In total, 0.5 μg of genomic DNA was digested with McrBc in the presence GTP (+). Samples lacking GTP served as the negative controls (−). F1R and F1G, pooled red-, and green-leafed segregants, respectively, from the 15F1 population; F1R1–11 and F1G1–11, 11 randomly selected red and green-leafed individuals, respectively, from the 15F1 population; +1, ATG translational start site.

    Article Snippet: The isolated DNA was then digested with 40 units of the methylation-sensitive restriction enzyme McrBC (New England Biolabs; M0272L) for 2 h, and the digestion buffer without GTPase was used as the negative control.

    Techniques: Methylation, Polymerase Chain Reaction