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    New England Biolabs hpych4iv
    Distinct DNA methylation profile between E11.5NSph and E14.5NSph. ( A ) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. ( B ) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. ( C ) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. ( D ) COBRA representing DNA methylation status of <t>HpyCH4IV</t> sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. ( E ) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.
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    Images

    1) Product Images from "DNA methylation profile dynamics of tissue-dependent and differentially methylated regions during mouse brain development"

    Article Title: DNA methylation profile dynamics of tissue-dependent and differentially methylated regions during mouse brain development

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-82

    Distinct DNA methylation profile between E11.5NSph and E14.5NSph. ( A ) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. ( B ) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. ( C ) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. ( D ) COBRA representing DNA methylation status of HpyCH4IV sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. ( E ) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.
    Figure Legend Snippet: Distinct DNA methylation profile between E11.5NSph and E14.5NSph. ( A ) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. ( B ) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. ( C ) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. ( D ) COBRA representing DNA methylation status of HpyCH4IV sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. ( E ) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.

    Techniques Used: DNA Methylation Assay, Cell Culture, Staining, Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, Methylation Sequencing, Methylation

    2) Product Images from "Risk Association of TOX3 and MMP7 Gene Polymorphisms with Sporadic Breast Cancer in Mexican Women"

    Article Title: Risk Association of TOX3 and MMP7 Gene Polymorphisms with Sporadic Breast Cancer in Mexican Women

    Journal: Current Oncology

    doi: 10.3390/curroncol29020086

    PCR-RFLPs results. Electrophoresis in 1.5% agarose gels of digested amplified products with Bpu10I and HpyCH4IV restriction enzymes (related to Figure S1 ) ( a ) Digested amplified products of SNP TOX3 rs3803662. ( b ) Digested amplified products of SNP MMP7 rs1943779. TT, CC, and CT genotypes are shown, and lane ML indicates the 100 to 1000 bp molecular gene ruler ladder.
    Figure Legend Snippet: PCR-RFLPs results. Electrophoresis in 1.5% agarose gels of digested amplified products with Bpu10I and HpyCH4IV restriction enzymes (related to Figure S1 ) ( a ) Digested amplified products of SNP TOX3 rs3803662. ( b ) Digested amplified products of SNP MMP7 rs1943779. TT, CC, and CT genotypes are shown, and lane ML indicates the 100 to 1000 bp molecular gene ruler ladder.

    Techniques Used: Polymerase Chain Reaction, Electrophoresis, Amplification

    3) Product Images from "Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells"

    Article Title: Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23822-4

    Methylation profile at major satellites. ( A ) Southern-blot analysis of gDNA digested with HpyCH4IV revealed with probe for major satellites. Linescan quantification for each lane: MEF (black), Dnmt TKO (pink), 2i-ESC (red), serum-ESC (blue) and EpiSC (green). ( B ) Western-blots for DNMT3A, DNMT3B and H3.
    Figure Legend Snippet: Methylation profile at major satellites. ( A ) Southern-blot analysis of gDNA digested with HpyCH4IV revealed with probe for major satellites. Linescan quantification for each lane: MEF (black), Dnmt TKO (pink), 2i-ESC (red), serum-ESC (blue) and EpiSC (green). ( B ) Western-blots for DNMT3A, DNMT3B and H3.

    Techniques Used: Methylation, Southern Blot, Western Blot

    4) Product Images from "DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) in mouse promoter regions demonstrating tissue-specific gene expression"

    Article Title: DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) in mouse promoter regions demonstrating tissue-specific gene expression

    Journal: Genome Research

    doi: 10.1101/gr.074070.107

    DNA methylation profiles were analyzed by D-REAM. ( A ) Illustration of the D-REAM method. Genomic DNA was digested with methylation-sensitive restriction enzyme HpyCH4IV and amplified by modified LM-PCR (Supplemental Fig. S1). Amplified fragments (gray bars) were hybridized with mouse promoter tiling array ( upper panel). Array signal intensities (vertical bars) were analyzed to identify regions corresponding to fragments in unmethylated HpyCH4IV loci. Comparison of signals from different samples enabled identification of differentially methylated regions ( lower panel). HpyCH4IV loci overlapping with regions yielding differential signals were defined as T-DMRtags. ( B ) Agarose gel electrophoresis of undigested (lane 2 ), HpyCH4IV-digested (lane 3 ), and HpyCH4IV–TaqI-digested (lane 4 ) mouse liver DNA. Positions corresponding to 0.1, 0.5, 1.0, and 2.0 kbp (lanes 1 , 5 ) are indicated on one side of the gel image. ( C ) Venn diagram of DNA methylation status at HpyCH4IV sites in mouse liver and cerebrum. Numbers without parentheses represent numbers of HpyCH4IV sites, while Ensembl transcripts IDs are in parentheses. Outer and inner rectangles represent whole mouse genome and regions covered by the promoter tiling array, respectively. Ovals indicate unmethylated HpyCH4IV sites of liver and cerebrum identified by D-REAM. ( D ) Correlation of microarray probe intensities in duplicate mouse liver experiments, plotted on logarithmic axes (base 2). ( E ) MATscore distribution of array regions corresponding to the TaqI–TaqI fragments (gray) and HpyCH4IV-digested fragments (black). The dotted line represents the MATscore cutoff value. ( F ) Reliability of comparative MAT analysis. Bar-plots of MATscores of the hypomethylated regions identified by MAT ( P
    Figure Legend Snippet: DNA methylation profiles were analyzed by D-REAM. ( A ) Illustration of the D-REAM method. Genomic DNA was digested with methylation-sensitive restriction enzyme HpyCH4IV and amplified by modified LM-PCR (Supplemental Fig. S1). Amplified fragments (gray bars) were hybridized with mouse promoter tiling array ( upper panel). Array signal intensities (vertical bars) were analyzed to identify regions corresponding to fragments in unmethylated HpyCH4IV loci. Comparison of signals from different samples enabled identification of differentially methylated regions ( lower panel). HpyCH4IV loci overlapping with regions yielding differential signals were defined as T-DMRtags. ( B ) Agarose gel electrophoresis of undigested (lane 2 ), HpyCH4IV-digested (lane 3 ), and HpyCH4IV–TaqI-digested (lane 4 ) mouse liver DNA. Positions corresponding to 0.1, 0.5, 1.0, and 2.0 kbp (lanes 1 , 5 ) are indicated on one side of the gel image. ( C ) Venn diagram of DNA methylation status at HpyCH4IV sites in mouse liver and cerebrum. Numbers without parentheses represent numbers of HpyCH4IV sites, while Ensembl transcripts IDs are in parentheses. Outer and inner rectangles represent whole mouse genome and regions covered by the promoter tiling array, respectively. Ovals indicate unmethylated HpyCH4IV sites of liver and cerebrum identified by D-REAM. ( D ) Correlation of microarray probe intensities in duplicate mouse liver experiments, plotted on logarithmic axes (base 2). ( E ) MATscore distribution of array regions corresponding to the TaqI–TaqI fragments (gray) and HpyCH4IV-digested fragments (black). The dotted line represents the MATscore cutoff value. ( F ) Reliability of comparative MAT analysis. Bar-plots of MATscores of the hypomethylated regions identified by MAT ( P

    Techniques Used: DNA Methylation Assay, Methylation, Amplification, Modification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Microarray

    Bisulfite sequencing of T-DMRs of liver-specific transcription factors, as Hnf1a ( A ), Hnf4a ( B ), Nr1h3 ( C ), Nr1i2 ( D ), and Rxra ( E ). Genomic structures are presented at the top of each figure section. The graphs in boxes toward the center in A and B represent CpG density (blue) and GC percentages (gray). The bars visible along the top of the center lines in A and B represent CpG dinucleotide positions; bars below represent HpyCH4IV sites. Boxes and arrowheads represent T-DMRs and T-DMRtags, respectively. IGB plots of comparative microarray signals corresponding to the regions in the abovementioned figures are displayed toward the bottom of the middle sections. Bisulfite sequencing data obtained for 10 isolates from liver (L) and cerebrum (C) are summarized at the bottom or side of the figure section. Open and closed circles represent unmethylated and methylated CpG, respectively.
    Figure Legend Snippet: Bisulfite sequencing of T-DMRs of liver-specific transcription factors, as Hnf1a ( A ), Hnf4a ( B ), Nr1h3 ( C ), Nr1i2 ( D ), and Rxra ( E ). Genomic structures are presented at the top of each figure section. The graphs in boxes toward the center in A and B represent CpG density (blue) and GC percentages (gray). The bars visible along the top of the center lines in A and B represent CpG dinucleotide positions; bars below represent HpyCH4IV sites. Boxes and arrowheads represent T-DMRs and T-DMRtags, respectively. IGB plots of comparative microarray signals corresponding to the regions in the abovementioned figures are displayed toward the bottom of the middle sections. Bisulfite sequencing data obtained for 10 isolates from liver (L) and cerebrum (C) are summarized at the bottom or side of the figure section. Open and closed circles represent unmethylated and methylated CpG, respectively.

    Techniques Used: Methylation Sequencing, Microarray, Methylation

    5) Product Images from "Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily"

    Article Title: Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily

    Journal: Mammalian genome : official journal of the International Mammalian Genome Society

    doi: 10.1007/s00335-011-9383-x

    DNA methylation status of PRL superfamily members in the placenta, liver, and cerebrum. a The upper panels show the integrated genome browser of comparative D-REAM signals. Arrowheads and vertical lines represent T-DMR and HpyCH4IV sites, respectively.
    Figure Legend Snippet: DNA methylation status of PRL superfamily members in the placenta, liver, and cerebrum. a The upper panels show the integrated genome browser of comparative D-REAM signals. Arrowheads and vertical lines represent T-DMR and HpyCH4IV sites, respectively.

    Techniques Used: DNA Methylation Assay

    6) Product Images from "Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks"

    Article Title: Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018844

    Epigenetic changes after SETD2 knockdown. A . Western blot for HBEC cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. B . Composite profile of epigenetic changes after SETD2 siRNA knockdown in HBEC. The profile was created for gene bodies marked by H3K36me3 and DNA methylation over at least 20% of gene body length. Each gene body was divided into 20 bins and the 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. C . Analysis of DNA methylation in the gene body of the NOTCH3 gene in conditions of H3K36me3 deficiency. The H3K36me3 profile of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfections in HBEC cells is shown. The region analyzed by COBRA methylation assays is indicated by a box. Using gene-specific primers, bisulfite-converted DNA was amplified. After cutting with HpyCH4IV recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were fractionated on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation. D . Analysis of H3K36me3 and DNA methylation in the gene body of RFX2 in conditions of H3K36me3 deficiency. The region analyzed by bisulfite sequencing is marked by a box. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 20 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences. E . Western blot for HCT116-DKO cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. F . DNA methylation analysis of the gene body of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfection of HCT116-DKO cells. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 13 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences.
    Figure Legend Snippet: Epigenetic changes after SETD2 knockdown. A . Western blot for HBEC cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. B . Composite profile of epigenetic changes after SETD2 siRNA knockdown in HBEC. The profile was created for gene bodies marked by H3K36me3 and DNA methylation over at least 20% of gene body length. Each gene body was divided into 20 bins and the 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. C . Analysis of DNA methylation in the gene body of the NOTCH3 gene in conditions of H3K36me3 deficiency. The H3K36me3 profile of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfections in HBEC cells is shown. The region analyzed by COBRA methylation assays is indicated by a box. Using gene-specific primers, bisulfite-converted DNA was amplified. After cutting with HpyCH4IV recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were fractionated on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation. D . Analysis of H3K36me3 and DNA methylation in the gene body of RFX2 in conditions of H3K36me3 deficiency. The region analyzed by bisulfite sequencing is marked by a box. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 20 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences. E . Western blot for HCT116-DKO cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. F . DNA methylation analysis of the gene body of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfection of HCT116-DKO cells. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 13 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences.

    Techniques Used: Western Blot, Transfection, DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, Methylation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, In Vitro, Positive Control, Methylation Sequencing, Clone Assay

    7) Product Images from "Selective amplification of hypermethylated DNA from diverse tumor types via MSRE-PCR"

    Article Title: Selective amplification of hypermethylated DNA from diverse tumor types via MSRE-PCR

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27825

    Methylation sensitive restriction sites in target cDMRs. Five MSREs are considered here: HhaI, HpaII, HpyCH4IV, AciI and BstUI. ( A ) Histogram of the number of MSRE sites in the target regions. ( B ) Scatterplot of the number of MSRE sites versus the number of CpGs in the target regions showing a linear relationship (R 2 = 0.89, p
    Figure Legend Snippet: Methylation sensitive restriction sites in target cDMRs. Five MSREs are considered here: HhaI, HpaII, HpyCH4IV, AciI and BstUI. ( A ) Histogram of the number of MSRE sites in the target regions. ( B ) Scatterplot of the number of MSRE sites versus the number of CpGs in the target regions showing a linear relationship (R 2 = 0.89, p

    Techniques Used: Methylation

    8) Product Images from "DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells"

    Article Title: DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-13-58

    UEA1 + CD45 − mTECs and UEA1 − CD45 − thymic stromal cells display distinct DNA methylation levels at mTECany2-T-DMRs. DNA methylation levels at the mTECany2-T-DMRs in mTECs and stromal cells isolated from Aire +/+ and Aire −/− littermate mice, as detected by COBRA. The levels of DNA methylation at CpGs within the HpyCH4IV site are indicated by the vertical axis. For Aire and CD80 genes—containing 2 HpyCH4IV sites inside the investigated region—the DNA methylation rate was calculated when any 1 of the CpGs was methylated. For the remaining genes, the levels represent DNA methylation levels at a single CpG. Bar graphs indicate means ± standard errors of the data from at least 3 independent bisulfite PCRs using independently isolated cells from 3 Aire +/+ mice and 3 Aire −/− mice.
    Figure Legend Snippet: UEA1 + CD45 − mTECs and UEA1 − CD45 − thymic stromal cells display distinct DNA methylation levels at mTECany2-T-DMRs. DNA methylation levels at the mTECany2-T-DMRs in mTECs and stromal cells isolated from Aire +/+ and Aire −/− littermate mice, as detected by COBRA. The levels of DNA methylation at CpGs within the HpyCH4IV site are indicated by the vertical axis. For Aire and CD80 genes—containing 2 HpyCH4IV sites inside the investigated region—the DNA methylation rate was calculated when any 1 of the CpGs was methylated. For the remaining genes, the levels represent DNA methylation levels at a single CpG. Bar graphs indicate means ± standard errors of the data from at least 3 independent bisulfite PCRs using independently isolated cells from 3 Aire +/+ mice and 3 Aire −/− mice.

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

    mTECany2-T-DMRs correlate with gene expression in mTECs and associate with marker genes of mTECs. A. Hierarchical clustering of D-REAM scores corresponding to mTECany2-T-DMRs identified by comparison of Aire −/− mTECs with the brain, thymus, liver, and ES cells by Pearson’s correlations. Among mTECany2-T-DMRs, those hypomethylated in Aire −/− mTECs in comparison with all of the reference cells and tissues were defined as mTECu-T-DMRs (indicated by asterisk). Br, brain; Thy, thymus; Lv, liver. B. Relative expression levels of genes associated with mTECu-T-DMRs in proximal regions (−6 to +2.5 kb) from TSS. The horizontal axis of the boxplot represents the log ratios of gene expression levels relative to Aire −/− mTECs. The numbers in round brackets indicate independent gene expression profiles for each type of cell or tissue (Additional file 5 : Table S2). The p -values of t -tests showing significant differences in relative expression levels are shown. C. Bisulfite sequencing of regions around the TSS of Aire . The arrowhead indicates the position of the mTECu-T-DMR. The regions upstream (−674 to −191 bp) and downstream (+1672 to +2082 bp) of the TSS were analyzed independently using bisulfite sequencing. Open and closed circles represent unmethylated and methylated CpGs, respectively. Positions of CpGs are shown as vertical bars above the genomic structure (shown at the bottom of the panel), and the boxes indicate exons. D. COBRA analysis of mTECu-T-DMRs associated with Cd80 and Trp63 . Positions of HpyCH4IV sites and mTECu-T-DMRs are shown above the genomic sequences within −1 to +2.5 kb of the TSS. Electropherograms represent the bisulfite PCR products after digestion with HpyCH4IV. Hypomethylated fragments were resistant to HpyCH4IV digestion (+). The levels of DNA methylation (%) are shown under each lane of the electropherograms.
    Figure Legend Snippet: mTECany2-T-DMRs correlate with gene expression in mTECs and associate with marker genes of mTECs. A. Hierarchical clustering of D-REAM scores corresponding to mTECany2-T-DMRs identified by comparison of Aire −/− mTECs with the brain, thymus, liver, and ES cells by Pearson’s correlations. Among mTECany2-T-DMRs, those hypomethylated in Aire −/− mTECs in comparison with all of the reference cells and tissues were defined as mTECu-T-DMRs (indicated by asterisk). Br, brain; Thy, thymus; Lv, liver. B. Relative expression levels of genes associated with mTECu-T-DMRs in proximal regions (−6 to +2.5 kb) from TSS. The horizontal axis of the boxplot represents the log ratios of gene expression levels relative to Aire −/− mTECs. The numbers in round brackets indicate independent gene expression profiles for each type of cell or tissue (Additional file 5 : Table S2). The p -values of t -tests showing significant differences in relative expression levels are shown. C. Bisulfite sequencing of regions around the TSS of Aire . The arrowhead indicates the position of the mTECu-T-DMR. The regions upstream (−674 to −191 bp) and downstream (+1672 to +2082 bp) of the TSS were analyzed independently using bisulfite sequencing. Open and closed circles represent unmethylated and methylated CpGs, respectively. Positions of CpGs are shown as vertical bars above the genomic structure (shown at the bottom of the panel), and the boxes indicate exons. D. COBRA analysis of mTECu-T-DMRs associated with Cd80 and Trp63 . Positions of HpyCH4IV sites and mTECu-T-DMRs are shown above the genomic sequences within −1 to +2.5 kb of the TSS. Electropherograms represent the bisulfite PCR products after digestion with HpyCH4IV. Hypomethylated fragments were resistant to HpyCH4IV digestion (+). The levels of DNA methylation (%) are shown under each lane of the electropherograms.

    Techniques Used: Expressing, Marker, Methylation Sequencing, Methylation, Combined Bisulfite Restriction Analysis Assay, Genomic Sequencing, Polymerase Chain Reaction, DNA Methylation Assay

    9) Product Images from "Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae"

    Article Title: Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-015-0580-8

    TRDMT1 and TRM4B methylate Arabidopsis nuclear encoded transfer RNAs. a Genomic origins of methylated and non-methylated tRNAs. Methylated tRNAs were only detected from the nuclear genome (3 biological replicates). b Above: clover-leaf representative secondary structure of tRNA indicating in red, the five cytosine positions methylated in wild type. Below: Heatmap showing percentage methylation of all cytosines detected in nuclear tRNAs of wild type, and mutants trdmt1 , trm4a , trm4b-1 and trdmt1 trm4b using RBS-seq. Cytosine positions are indicated next to tRNA isodecoders. White boxes represent cytosine positions with coverage less than five reads. (wild type 3 biological replicates, mutants n = 1). c Genomic structure of trm4a and trm4b mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by TRDMT1 at position C38 on BS treated tRNA Asp(GTC) template. Above: Restriction maps of PCR amplified products showing the expected digest patterns of methylated and non-methylated template. Below: Cleavage of PCR amplified product by HpyCH4IV confirms C38 methylation in wild type as opposed to non-methylated C38 in trdmt1 results in loss of HpyCH4IV restriction site. Loading control is undigested PCR product. e Hygromycin B stress assay. Trdmt1 trm4b double mutants and to a lesser extent, trm4b-1 mutants display increased sensitivity to hygromycin B (Hyg) at 10 and 20 days after germination (DAG) compared to controls
    Figure Legend Snippet: TRDMT1 and TRM4B methylate Arabidopsis nuclear encoded transfer RNAs. a Genomic origins of methylated and non-methylated tRNAs. Methylated tRNAs were only detected from the nuclear genome (3 biological replicates). b Above: clover-leaf representative secondary structure of tRNA indicating in red, the five cytosine positions methylated in wild type. Below: Heatmap showing percentage methylation of all cytosines detected in nuclear tRNAs of wild type, and mutants trdmt1 , trm4a , trm4b-1 and trdmt1 trm4b using RBS-seq. Cytosine positions are indicated next to tRNA isodecoders. White boxes represent cytosine positions with coverage less than five reads. (wild type 3 biological replicates, mutants n = 1). c Genomic structure of trm4a and trm4b mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by TRDMT1 at position C38 on BS treated tRNA Asp(GTC) template. Above: Restriction maps of PCR amplified products showing the expected digest patterns of methylated and non-methylated template. Below: Cleavage of PCR amplified product by HpyCH4IV confirms C38 methylation in wild type as opposed to non-methylated C38 in trdmt1 results in loss of HpyCH4IV restriction site. Loading control is undigested PCR product. e Hygromycin B stress assay. Trdmt1 trm4b double mutants and to a lesser extent, trm4b-1 mutants display increased sensitivity to hygromycin B (Hyg) at 10 and 20 days after germination (DAG) compared to controls

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification

    10) Product Images from "NuRD-dependent DNA methylation prevents ES cells from accessing a trophectoderm fate"

    Article Title: NuRD-dependent DNA methylation prevents ES cells from accessing a trophectoderm fate

    Journal: Biology Open

    doi: 10.1242/bio.2012513

    Global analysis of DNA methylation in Mbd3 −/− ES cells. Genomic DNA from parental ES cells (+/−), Mbd3 −/− ES cells (−/−) or from ES cells expressing only Mbd3a, Mbd3b or Mbd3c was digested with MspI (M) and HpaII (H) (A) or with HpyCH4IV (B) before being Southern blotted and hybridised with probes for the minor (A) or major satellite DNA repeats (B), or for IAP LTRs (A). Mito: mitochondrial DNA probe used as a loading and digestion control. (C) Total 5-methylcytosine levels were quantitated in two different Mbd3 Flox/- ES cell lines (WT1 and WT2) and two different Mbd3 −/− ES cell lines (KO1 and KO2) by HPLC and mass spectrometry. Error bars represent SEM of three technical replicates.
    Figure Legend Snippet: Global analysis of DNA methylation in Mbd3 −/− ES cells. Genomic DNA from parental ES cells (+/−), Mbd3 −/− ES cells (−/−) or from ES cells expressing only Mbd3a, Mbd3b or Mbd3c was digested with MspI (M) and HpaII (H) (A) or with HpyCH4IV (B) before being Southern blotted and hybridised with probes for the minor (A) or major satellite DNA repeats (B), or for IAP LTRs (A). Mito: mitochondrial DNA probe used as a loading and digestion control. (C) Total 5-methylcytosine levels were quantitated in two different Mbd3 Flox/- ES cell lines (WT1 and WT2) and two different Mbd3 −/− ES cell lines (KO1 and KO2) by HPLC and mass spectrometry. Error bars represent SEM of three technical replicates.

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

    11) Product Images from "DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells"

    Article Title: DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-13-58

    UEA1 + CD45 − mTECs and UEA1 − CD45 − thymic stromal cells display distinct DNA methylation levels at mTECany2-T-DMRs. DNA methylation levels at the mTECany2-T-DMRs in mTECs and stromal cells isolated from Aire +/+ and Aire −/− littermate mice, as detected by COBRA. The levels of DNA methylation at CpGs within the HpyCH4IV site are indicated by the vertical axis. For Aire and CD80 genes—containing 2 HpyCH4IV sites inside the investigated region—the DNA methylation rate was calculated when any 1 of the CpGs was methylated. For the remaining genes, the levels represent DNA methylation levels at a single CpG. Bar graphs indicate means ± standard errors of the data from at least 3 independent bisulfite PCRs using independently isolated cells from 3 Aire +/+ mice and 3 Aire −/− mice.
    Figure Legend Snippet: UEA1 + CD45 − mTECs and UEA1 − CD45 − thymic stromal cells display distinct DNA methylation levels at mTECany2-T-DMRs. DNA methylation levels at the mTECany2-T-DMRs in mTECs and stromal cells isolated from Aire +/+ and Aire −/− littermate mice, as detected by COBRA. The levels of DNA methylation at CpGs within the HpyCH4IV site are indicated by the vertical axis. For Aire and CD80 genes—containing 2 HpyCH4IV sites inside the investigated region—the DNA methylation rate was calculated when any 1 of the CpGs was methylated. For the remaining genes, the levels represent DNA methylation levels at a single CpG. Bar graphs indicate means ± standard errors of the data from at least 3 independent bisulfite PCRs using independently isolated cells from 3 Aire +/+ mice and 3 Aire −/− mice.

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

    mTECany2-T-DMRs correlate with gene expression in mTECs and associate with marker genes of mTECs. A. Hierarchical clustering of D-REAM scores corresponding to mTECany2-T-DMRs identified by comparison of Aire −/− mTECs with the brain, thymus, liver, and ES cells by Pearson’s correlations. Among mTECany2-T-DMRs, those hypomethylated in Aire −/− mTECs in comparison with all of the reference cells and tissues were defined as mTECu-T-DMRs (indicated by asterisk). Br, brain; Thy, thymus; Lv, liver. B. Relative expression levels of genes associated with mTECu-T-DMRs in proximal regions (−6 to +2.5 kb) from TSS. The horizontal axis of the boxplot represents the log ratios of gene expression levels relative to Aire −/− mTECs. The numbers in round brackets indicate independent gene expression profiles for each type of cell or tissue (Additional file 5 : Table S2). The p -values of t -tests showing significant differences in relative expression levels are shown. C. Bisulfite sequencing of regions around the TSS of Aire . The arrowhead indicates the position of the mTECu-T-DMR. The regions upstream (−674 to −191 bp) and downstream (+1672 to +2082 bp) of the TSS were analyzed independently using bisulfite sequencing. Open and closed circles represent unmethylated and methylated CpGs, respectively. Positions of CpGs are shown as vertical bars above the genomic structure (shown at the bottom of the panel), and the boxes indicate exons. D. COBRA analysis of mTECu-T-DMRs associated with Cd80 and Trp63 . Positions of HpyCH4IV sites and mTECu-T-DMRs are shown above the genomic sequences within −1 to +2.5 kb of the TSS. Electropherograms represent the bisulfite PCR products after digestion with HpyCH4IV. Hypomethylated fragments were resistant to HpyCH4IV digestion (+). The levels of DNA methylation (%) are shown under each lane of the electropherograms.
    Figure Legend Snippet: mTECany2-T-DMRs correlate with gene expression in mTECs and associate with marker genes of mTECs. A. Hierarchical clustering of D-REAM scores corresponding to mTECany2-T-DMRs identified by comparison of Aire −/− mTECs with the brain, thymus, liver, and ES cells by Pearson’s correlations. Among mTECany2-T-DMRs, those hypomethylated in Aire −/− mTECs in comparison with all of the reference cells and tissues were defined as mTECu-T-DMRs (indicated by asterisk). Br, brain; Thy, thymus; Lv, liver. B. Relative expression levels of genes associated with mTECu-T-DMRs in proximal regions (−6 to +2.5 kb) from TSS. The horizontal axis of the boxplot represents the log ratios of gene expression levels relative to Aire −/− mTECs. The numbers in round brackets indicate independent gene expression profiles for each type of cell or tissue (Additional file 5 : Table S2). The p -values of t -tests showing significant differences in relative expression levels are shown. C. Bisulfite sequencing of regions around the TSS of Aire . The arrowhead indicates the position of the mTECu-T-DMR. The regions upstream (−674 to −191 bp) and downstream (+1672 to +2082 bp) of the TSS were analyzed independently using bisulfite sequencing. Open and closed circles represent unmethylated and methylated CpGs, respectively. Positions of CpGs are shown as vertical bars above the genomic structure (shown at the bottom of the panel), and the boxes indicate exons. D. COBRA analysis of mTECu-T-DMRs associated with Cd80 and Trp63 . Positions of HpyCH4IV sites and mTECu-T-DMRs are shown above the genomic sequences within −1 to +2.5 kb of the TSS. Electropherograms represent the bisulfite PCR products after digestion with HpyCH4IV. Hypomethylated fragments were resistant to HpyCH4IV digestion (+). The levels of DNA methylation (%) are shown under each lane of the electropherograms.

    Techniques Used: Expressing, Marker, Methylation Sequencing, Methylation, Combined Bisulfite Restriction Analysis Assay, Genomic Sequencing, Polymerase Chain Reaction, DNA Methylation Assay

    12) Product Images from "Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily"

    Article Title: Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily

    Journal: Mammalian genome : official journal of the International Mammalian Genome Society

    doi: 10.1007/s00335-011-9383-x

    DNA methylation status of PRL superfamily members in the placenta, liver, and cerebrum. a The upper panels show the integrated genome browser of comparative D-REAM signals. Arrowheads and vertical lines represent T-DMR and HpyCH4IV sites, respectively.
    Figure Legend Snippet: DNA methylation status of PRL superfamily members in the placenta, liver, and cerebrum. a The upper panels show the integrated genome browser of comparative D-REAM signals. Arrowheads and vertical lines represent T-DMR and HpyCH4IV sites, respectively.

    Techniques Used: DNA Methylation Assay

    13) Product Images from "Loss of Function of the E1-Like-b Gene Associates With Early Flowering Under Long-Day Conditions in Soybean"

    Article Title: Loss of Function of the E1-Like-b Gene Associates With Early Flowering Under Long-Day Conditions in Soybean

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01867

    Derived cleaved amplified polymorphic sequence (dCAPS) marker analysis to detect the single-base deletion in the e1lb allele. (A) Primers designed and the generation of an HpyCH4IV restriction site. One of three cytosines marked by red was deleted in ZE. (B) Gel electrophoresis of PCR products without (–) or with (+) HpyCH4IV digestion. H- e3 , Harosoy NIL for e3 ; ZE, Zeika.
    Figure Legend Snippet: Derived cleaved amplified polymorphic sequence (dCAPS) marker analysis to detect the single-base deletion in the e1lb allele. (A) Primers designed and the generation of an HpyCH4IV restriction site. One of three cytosines marked by red was deleted in ZE. (B) Gel electrophoresis of PCR products without (–) or with (+) HpyCH4IV digestion. H- e3 , Harosoy NIL for e3 ; ZE, Zeika.

    Techniques Used: Derivative Assay, Amplification, Sequencing, Marker, Nucleic Acid Electrophoresis, Polymerase Chain Reaction

    14) Product Images from "Loss of Function of the E1-Like-b Gene Associates With Early Flowering Under Long-Day Conditions in Soybean"

    Article Title: Loss of Function of the E1-Like-b Gene Associates With Early Flowering Under Long-Day Conditions in Soybean

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01867

    Derived cleaved amplified polymorphic sequence (dCAPS) marker analysis to detect the single-base deletion in the e1lb allele. (A) Primers designed and the generation of an HpyCH4IV restriction site. One of three cytosines marked by red was deleted in ZE. (B) Gel electrophoresis of PCR products without (–) or with (+) HpyCH4IV digestion. H- e3 , Harosoy NIL for e3 ; ZE, Zeika.
    Figure Legend Snippet: Derived cleaved amplified polymorphic sequence (dCAPS) marker analysis to detect the single-base deletion in the e1lb allele. (A) Primers designed and the generation of an HpyCH4IV restriction site. One of three cytosines marked by red was deleted in ZE. (B) Gel electrophoresis of PCR products without (–) or with (+) HpyCH4IV digestion. H- e3 , Harosoy NIL for e3 ; ZE, Zeika.

    Techniques Used: Derivative Assay, Amplification, Sequencing, Marker, Nucleic Acid Electrophoresis, Polymerase Chain Reaction

    15) Product Images from "DNA methylation profile dynamics of tissue-dependent and differentially methylated regions during mouse brain development"

    Article Title: DNA methylation profile dynamics of tissue-dependent and differentially methylated regions during mouse brain development

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-82

    Distinct DNA methylation profile between E11.5NSph and E14.5NSph. ( A ) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. ( B ) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. ( C ) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. ( D ) COBRA representing DNA methylation status of HpyCH4IV sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. ( E ) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.
    Figure Legend Snippet: Distinct DNA methylation profile between E11.5NSph and E14.5NSph. ( A ) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. ( B ) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. ( C ) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. ( D ) COBRA representing DNA methylation status of HpyCH4IV sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. ( E ) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.

    Techniques Used: DNA Methylation Assay, Cell Culture, Staining, Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, Methylation Sequencing, Methylation

    16) Product Images from "DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) in mouse promoter regions demonstrating tissue-specific gene expression"

    Article Title: DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) in mouse promoter regions demonstrating tissue-specific gene expression

    Journal: Genome Research

    doi: 10.1101/gr.074070.107

    DNA methylation profiles were analyzed by D-REAM. ( A ) Illustration of the D-REAM method. Genomic DNA was digested with methylation-sensitive restriction enzyme HpyCH4IV and amplified by modified LM-PCR (Supplemental Fig. S1). Amplified fragments (gray bars) were hybridized with mouse promoter tiling array ( upper panel). Array signal intensities (vertical bars) were analyzed to identify regions corresponding to fragments in unmethylated HpyCH4IV loci. Comparison of signals from different samples enabled identification of differentially methylated regions ( lower panel). HpyCH4IV loci overlapping with regions yielding differential signals were defined as T-DMRtags. ( B ) Agarose gel electrophoresis of undigested (lane 2 ), HpyCH4IV-digested (lane 3 ), and HpyCH4IV–TaqI-digested (lane 4 ) mouse liver DNA. Positions corresponding to 0.1, 0.5, 1.0, and 2.0 kbp (lanes 1 , 5 ) are indicated on one side of the gel image. ( C ) Venn diagram of DNA methylation status at HpyCH4IV sites in mouse liver and cerebrum. Numbers without parentheses represent numbers of HpyCH4IV sites, while Ensembl transcripts IDs are in parentheses. Outer and inner rectangles represent whole mouse genome and regions covered by the promoter tiling array, respectively. Ovals indicate unmethylated HpyCH4IV sites of liver and cerebrum identified by D-REAM. ( D ) Correlation of microarray probe intensities in duplicate mouse liver experiments, plotted on logarithmic axes (base 2). ( E ) MATscore distribution of array regions corresponding to the TaqI–TaqI fragments (gray) and HpyCH4IV-digested fragments (black). The dotted line represents the MATscore cutoff value. ( F ) Reliability of comparative MAT analysis. Bar-plots of MATscores of the hypomethylated regions identified by MAT ( P
    Figure Legend Snippet: DNA methylation profiles were analyzed by D-REAM. ( A ) Illustration of the D-REAM method. Genomic DNA was digested with methylation-sensitive restriction enzyme HpyCH4IV and amplified by modified LM-PCR (Supplemental Fig. S1). Amplified fragments (gray bars) were hybridized with mouse promoter tiling array ( upper panel). Array signal intensities (vertical bars) were analyzed to identify regions corresponding to fragments in unmethylated HpyCH4IV loci. Comparison of signals from different samples enabled identification of differentially methylated regions ( lower panel). HpyCH4IV loci overlapping with regions yielding differential signals were defined as T-DMRtags. ( B ) Agarose gel electrophoresis of undigested (lane 2 ), HpyCH4IV-digested (lane 3 ), and HpyCH4IV–TaqI-digested (lane 4 ) mouse liver DNA. Positions corresponding to 0.1, 0.5, 1.0, and 2.0 kbp (lanes 1 , 5 ) are indicated on one side of the gel image. ( C ) Venn diagram of DNA methylation status at HpyCH4IV sites in mouse liver and cerebrum. Numbers without parentheses represent numbers of HpyCH4IV sites, while Ensembl transcripts IDs are in parentheses. Outer and inner rectangles represent whole mouse genome and regions covered by the promoter tiling array, respectively. Ovals indicate unmethylated HpyCH4IV sites of liver and cerebrum identified by D-REAM. ( D ) Correlation of microarray probe intensities in duplicate mouse liver experiments, plotted on logarithmic axes (base 2). ( E ) MATscore distribution of array regions corresponding to the TaqI–TaqI fragments (gray) and HpyCH4IV-digested fragments (black). The dotted line represents the MATscore cutoff value. ( F ) Reliability of comparative MAT analysis. Bar-plots of MATscores of the hypomethylated regions identified by MAT ( P

    Techniques Used: DNA Methylation Assay, Methylation, Amplification, Modification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Microarray

    Bisulfite sequencing of T-DMRs of liver-specific transcription factors, as Hnf1a ( A ), Hnf4a ( B ), Nr1h3 ( C ), Nr1i2 ( D ), and Rxra ( E ). Genomic structures are presented at the top of each figure section. The graphs in boxes toward the center in A and B represent CpG density (blue) and GC percentages (gray). The bars visible along the top of the center lines in A and B represent CpG dinucleotide positions; bars below represent HpyCH4IV sites. Boxes and arrowheads represent T-DMRs and T-DMRtags, respectively. IGB plots of comparative microarray signals corresponding to the regions in the abovementioned figures are displayed toward the bottom of the middle sections. Bisulfite sequencing data obtained for 10 isolates from liver (L) and cerebrum (C) are summarized at the bottom or side of the figure section. Open and closed circles represent unmethylated and methylated CpG, respectively.
    Figure Legend Snippet: Bisulfite sequencing of T-DMRs of liver-specific transcription factors, as Hnf1a ( A ), Hnf4a ( B ), Nr1h3 ( C ), Nr1i2 ( D ), and Rxra ( E ). Genomic structures are presented at the top of each figure section. The graphs in boxes toward the center in A and B represent CpG density (blue) and GC percentages (gray). The bars visible along the top of the center lines in A and B represent CpG dinucleotide positions; bars below represent HpyCH4IV sites. Boxes and arrowheads represent T-DMRs and T-DMRtags, respectively. IGB plots of comparative microarray signals corresponding to the regions in the abovementioned figures are displayed toward the bottom of the middle sections. Bisulfite sequencing data obtained for 10 isolates from liver (L) and cerebrum (C) are summarized at the bottom or side of the figure section. Open and closed circles represent unmethylated and methylated CpG, respectively.

    Techniques Used: Methylation Sequencing, Microarray, Methylation

    17) Product Images from "Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences"

    Article Title: Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-264

    Methylation of RASSFs, MSTs and WW45 in thyroid cancer cell lines . The methylation status of the indicated CpG islands was analyzed by COBRA. PCR products (for length see Additional file 5 , Table S1) from bisulfite-treated DNA were digested (+) and mock digested (-) with TaqI or HpyCH4IV and resolved on 2% gel. In vitro methylated DNA (methy.) was utilized as positive control. The sizes of a 100 bp ladder (M) are indicated. Methylated (m), partially methylated (p) and unmethylated (u) samples were determined.
    Figure Legend Snippet: Methylation of RASSFs, MSTs and WW45 in thyroid cancer cell lines . The methylation status of the indicated CpG islands was analyzed by COBRA. PCR products (for length see Additional file 5 , Table S1) from bisulfite-treated DNA were digested (+) and mock digested (-) with TaqI or HpyCH4IV and resolved on 2% gel. In vitro methylated DNA (methy.) was utilized as positive control. The sizes of a 100 bp ladder (M) are indicated. Methylated (m), partially methylated (p) and unmethylated (u) samples were determined.

    Techniques Used: Methylation, Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, In Vitro, Positive Control

    18) Product Images from "BDNF rs6265 Polymorphism and Its Methylation in Patients with Stroke Undergoing Rehabilitation"

    Article Title: BDNF rs6265 Polymorphism and Its Methylation in Patients with Stroke Undergoing Rehabilitation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21228438

    Brain Derived Neurotrophic Factor ( BDNF) rs6265 polymorphism genotyping. Upper panel: schematic representation of human BDNF exon IX (the roman numerals indicate the exon number). The translational (ATG) start site is indicated as an arrow and the region containing rs6265 SNP as a black box. Lower panel: the BDNF sequence amplified by PCR using BD-F and BD-R are indicated as arrows [ 36 ] while the restriction fragments of HpyCH4IV restriction enzyme as dashed lines. Lower panel: Electrophoresis on 2% agarose gels of PCR fragments after HpyCH4IV digestion. One PCR product of 84 bp indicated homozygous A/A allele, three PCR fragments of 84 bp, 56 bp and 28 bp indicated heterozygous A/G and two PCR products of 56 bp and 28 bp indicated homozygous G/G allele. The size of molecular the weight (MW) markers of the DNA ladder are in the left column.
    Figure Legend Snippet: Brain Derived Neurotrophic Factor ( BDNF) rs6265 polymorphism genotyping. Upper panel: schematic representation of human BDNF exon IX (the roman numerals indicate the exon number). The translational (ATG) start site is indicated as an arrow and the region containing rs6265 SNP as a black box. Lower panel: the BDNF sequence amplified by PCR using BD-F and BD-R are indicated as arrows [ 36 ] while the restriction fragments of HpyCH4IV restriction enzyme as dashed lines. Lower panel: Electrophoresis on 2% agarose gels of PCR fragments after HpyCH4IV digestion. One PCR product of 84 bp indicated homozygous A/A allele, three PCR fragments of 84 bp, 56 bp and 28 bp indicated heterozygous A/G and two PCR products of 56 bp and 28 bp indicated homozygous G/G allele. The size of molecular the weight (MW) markers of the DNA ladder are in the left column.

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

    19) Product Images from "Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily"

    Article Title: Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily

    Journal: Mammalian genome : official journal of the International Mammalian Genome Society

    doi: 10.1007/s00335-011-9383-x

    DNA methylation status of PRL superfamily members in the placenta, liver, and cerebrum. a The upper panels show the integrated genome browser of comparative D-REAM signals. Arrowheads and vertical lines represent T-DMR and HpyCH4IV sites, respectively.
    Figure Legend Snippet: DNA methylation status of PRL superfamily members in the placenta, liver, and cerebrum. a The upper panels show the integrated genome browser of comparative D-REAM signals. Arrowheads and vertical lines represent T-DMR and HpyCH4IV sites, respectively.

    Techniques Used: DNA Methylation Assay

    20) Product Images from "DICER1 Expression and Outcomes in Endometrioid Endometrial Adenocarcinoma"

    Article Title: DICER1 Expression and Outcomes in Endometrioid Endometrial Adenocarcinoma

    Journal: Cancer

    doi: 10.1002/cncr.25665

    Loss of heterozygosity analyses Panel A: PCR amplification products of marker rs1057035 in representative normal (N) / tumor (T) pairs digested with MwoI. Tumor 1870 demonstrates LOH (DICER1 expression = 2.38 arbitrary units). Panel B: PCR amplification products of marker rs1209904 in representative normal (N) / tumor (T) pairs digested with HpyCH4IV. Tumors 1641 (DICER1 expression = 12.29 arbitrary units), 1870 (DICER1 expression = 2.38 arbitrary units) and 1907 (DICER1 expression = 0.48 arbitrary units) demonstrate allelic imbalance. White arrows indicate the allelic fragment(s) with reduced intensity.
    Figure Legend Snippet: Loss of heterozygosity analyses Panel A: PCR amplification products of marker rs1057035 in representative normal (N) / tumor (T) pairs digested with MwoI. Tumor 1870 demonstrates LOH (DICER1 expression = 2.38 arbitrary units). Panel B: PCR amplification products of marker rs1209904 in representative normal (N) / tumor (T) pairs digested with HpyCH4IV. Tumors 1641 (DICER1 expression = 12.29 arbitrary units), 1870 (DICER1 expression = 2.38 arbitrary units) and 1907 (DICER1 expression = 0.48 arbitrary units) demonstrate allelic imbalance. White arrows indicate the allelic fragment(s) with reduced intensity.

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Expressing

    21) Product Images from "Nonalternating purine pyrimidine sequences can form stable left-handed DNA duplex by strong topological constraint"

    Article Title: Nonalternating purine pyrimidine sequences can form stable left-handed DNA duplex by strong topological constraint

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab1283

    Analysis of the positions of left-handed DNA in the LR-chimeras (8% PAGE). ( A ) Cleavage (5 min) of lc-74 0 and cc-74 0 . Lane 2: lc-74 0 ; lanes 3 and 4: lc-74 0 cleaved by SphI and MobI, respectively; lane 5: cc-74 0 ; lanes 6,7: cc-74 0 cleaved by SphI and MobI, respectively. ( B ) Schematic illustration of lc-74 0 and linear products of cleavage by SphI and MobI, respectively. ( C ) Cleavage (5 min) of lc-74 1 and cc-74 1 . Lane 2: lc-74 1 ; lanes 3–5: lc-74 1 cleaved by EcoRI, MobI, and Hpych4Iv, respectively; lane 6: cc-74 1 ; lanes 7–9: cc-74 1 cleaved by EcoRI, MobI, and Hpych4Iv, respectively. ( D ) Schematic illustration of lc-74 1 , lc-74 1EcoRI and linear products of cleavage by EcoRI, MobI, and Hpych4Iv, respectively. ( E ) Cleavage (30 min) of cc-74 0 and cc-74 1 after binding to Z22. Lane 1: Z22 bind to cc-74 0 ; lanes 2, 3: Cleavage of cc-74 0 by SphI and MboI after binding to Z22, respectively; lane 4: Z22 bind to cc-74 1 ; lanes 5–7: Cleavage of cc-74 1 by EcoRI, MboI, and Hpych4Iv after binding to Z22, respectively.
    Figure Legend Snippet: Analysis of the positions of left-handed DNA in the LR-chimeras (8% PAGE). ( A ) Cleavage (5 min) of lc-74 0 and cc-74 0 . Lane 2: lc-74 0 ; lanes 3 and 4: lc-74 0 cleaved by SphI and MobI, respectively; lane 5: cc-74 0 ; lanes 6,7: cc-74 0 cleaved by SphI and MobI, respectively. ( B ) Schematic illustration of lc-74 0 and linear products of cleavage by SphI and MobI, respectively. ( C ) Cleavage (5 min) of lc-74 1 and cc-74 1 . Lane 2: lc-74 1 ; lanes 3–5: lc-74 1 cleaved by EcoRI, MobI, and Hpych4Iv, respectively; lane 6: cc-74 1 ; lanes 7–9: cc-74 1 cleaved by EcoRI, MobI, and Hpych4Iv, respectively. ( D ) Schematic illustration of lc-74 1 , lc-74 1EcoRI and linear products of cleavage by EcoRI, MobI, and Hpych4Iv, respectively. ( E ) Cleavage (30 min) of cc-74 0 and cc-74 1 after binding to Z22. Lane 1: Z22 bind to cc-74 0 ; lanes 2, 3: Cleavage of cc-74 0 by SphI and MboI after binding to Z22, respectively; lane 4: Z22 bind to cc-74 1 ; lanes 5–7: Cleavage of cc-74 1 by EcoRI, MboI, and Hpych4Iv after binding to Z22, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Binding Assay

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    New England Biolabs hpych4iv
    Distinct DNA methylation profile between E11.5NSph and E14.5NSph. ( A ) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. ( B ) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. ( C ) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. ( D ) COBRA representing DNA methylation status of <t>HpyCH4IV</t> sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. ( E ) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.
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    Distinct DNA methylation profile between E11.5NSph and E14.5NSph. ( A ) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. ( B ) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. ( C ) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. ( D ) COBRA representing DNA methylation status of HpyCH4IV sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. ( E ) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.

    Journal: BMC Genomics

    Article Title: DNA methylation profile dynamics of tissue-dependent and differentially methylated regions during mouse brain development

    doi: 10.1186/1471-2164-14-82

    Figure Lengend Snippet: Distinct DNA methylation profile between E11.5NSph and E14.5NSph. ( A ) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. ( B ) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. ( C ) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. ( D ) COBRA representing DNA methylation status of HpyCH4IV sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. ( E ) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.

    Article Snippet: For COBRA, one-tenth of the PCR product was digested with HpyCH4IV at 37°C overnight and electrophoresed with untreated control in a 2% agarose gel.

    Techniques: DNA Methylation Assay, Cell Culture, Staining, Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, Methylation Sequencing, Methylation

    PCR-RFLPs results. Electrophoresis in 1.5% agarose gels of digested amplified products with Bpu10I and HpyCH4IV restriction enzymes (related to Figure S1 ) ( a ) Digested amplified products of SNP TOX3 rs3803662. ( b ) Digested amplified products of SNP MMP7 rs1943779. TT, CC, and CT genotypes are shown, and lane ML indicates the 100 to 1000 bp molecular gene ruler ladder.

    Journal: Current Oncology

    Article Title: Risk Association of TOX3 and MMP7 Gene Polymorphisms with Sporadic Breast Cancer in Mexican Women

    doi: 10.3390/curroncol29020086

    Figure Lengend Snippet: PCR-RFLPs results. Electrophoresis in 1.5% agarose gels of digested amplified products with Bpu10I and HpyCH4IV restriction enzymes (related to Figure S1 ) ( a ) Digested amplified products of SNP TOX3 rs3803662. ( b ) Digested amplified products of SNP MMP7 rs1943779. TT, CC, and CT genotypes are shown, and lane ML indicates the 100 to 1000 bp molecular gene ruler ladder.

    Article Snippet: The amplified products of the TOX3 rs3803662 and MMP7 rs1943779 polymorphisms were digested with 2.5 U of Bpu10I and 5 U of HpyCH4IV, respectively (New England Biolabs, Beverly, MA, USA).

    Techniques: Polymerase Chain Reaction, Electrophoresis, Amplification

    Workflow of cDNA-AFLP. Outline of the cDNA-AFLP procedure using the Bst143I and HpyCH4IV restriction enzyme combination: (i) mRNA is converted into double-stranded cDNA using a biotinylated oligo-dT primer. cDNA molecules are immobilized via the biotin tag to a streptavidin-coated reaction tube. (ii) First digestion of cDNA with Bst143I (indicated by gray arrow) and washing off the mobilized cDNA fragments. (iii) Second digestion of immobilized cDNA fragments with HpyCH4IV (indicated by gray arrow) and collection of mobilized cDNA fragments. These are used as template for (iv) ligation of DNA adapter to the restriction sites overhang. (v) Non-selective PCR amplification of cDNA fragments using primers (indicated by black arrows) compatible to adapter sequences. (vi) Final selective amplification of subsets of cDNA fragments using Bst143I+A and HpyCH4IV+NNN primers representing four selective nucleotides, with the Bst143I primer being labeled with the fluorescent dye Cy5 to allow subsequent detection of the cDNA fragments; and (vii) electrophoretic size fractionation and display on denaturing polyacrylamide gels of the Bst143I/HpyCH4IV cDNA fragments.

    Journal: Electrophoresis

    Article Title: Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP)

    doi: 10.1002/elps.200800756

    Figure Lengend Snippet: Workflow of cDNA-AFLP. Outline of the cDNA-AFLP procedure using the Bst143I and HpyCH4IV restriction enzyme combination: (i) mRNA is converted into double-stranded cDNA using a biotinylated oligo-dT primer. cDNA molecules are immobilized via the biotin tag to a streptavidin-coated reaction tube. (ii) First digestion of cDNA with Bst143I (indicated by gray arrow) and washing off the mobilized cDNA fragments. (iii) Second digestion of immobilized cDNA fragments with HpyCH4IV (indicated by gray arrow) and collection of mobilized cDNA fragments. These are used as template for (iv) ligation of DNA adapter to the restriction sites overhang. (v) Non-selective PCR amplification of cDNA fragments using primers (indicated by black arrows) compatible to adapter sequences. (vi) Final selective amplification of subsets of cDNA fragments using Bst143I+A and HpyCH4IV+NNN primers representing four selective nucleotides, with the Bst143I primer being labeled with the fluorescent dye Cy5 to allow subsequent detection of the cDNA fragments; and (vii) electrophoretic size fractionation and display on denaturing polyacrylamide gels of the Bst143I/HpyCH4IV cDNA fragments.

    Article Snippet: Truncated cDNA template bound to the column was digested with restriction endonuclease HpyCH4IV (isoschisomer of MaeII, New England Biolabs, Beverly, MA, USA).

    Techniques: cDNA-AFLP Assay, Ligation, Polymerase Chain Reaction, Amplification, Labeling, Fractionation

    Methylation profile at major satellites. ( A ) Southern-blot analysis of gDNA digested with HpyCH4IV revealed with probe for major satellites. Linescan quantification for each lane: MEF (black), Dnmt TKO (pink), 2i-ESC (red), serum-ESC (blue) and EpiSC (green). ( B ) Western-blots for DNMT3A, DNMT3B and H3.

    Journal: Scientific Reports

    Article Title: Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells

    doi: 10.1038/s41598-018-23822-4

    Figure Lengend Snippet: Methylation profile at major satellites. ( A ) Southern-blot analysis of gDNA digested with HpyCH4IV revealed with probe for major satellites. Linescan quantification for each lane: MEF (black), Dnmt TKO (pink), 2i-ESC (red), serum-ESC (blue) and EpiSC (green). ( B ) Western-blots for DNMT3A, DNMT3B and H3.

    Article Snippet: For major satellite analysis 200 ng of genomic DNA were digested with HpyCH4IV (New England Biolabs) for 1h at 37 °C, while for minor satellites 500ng of gDNA were digested with HpaII (New England Biolabs) and 300 ng with MspI (New England Biolabs), both O/N at 37 °C.

    Techniques: Methylation, Southern Blot, Western Blot