gpc methyltransferase treatment  (New England Biolabs)


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    New England Biolabs gpc methyltransferase treatment
    DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the <t>methyltransferase</t> M.CviPI to measure CA in the <t>GpC</t> dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.
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    Images

    1) Product Images from "Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation"

    Article Title: Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation

    Journal: bioRxiv

    doi: 10.1101/2022.05.19.492653

    DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the methyltransferase M.CviPI to measure CA in the GpC dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.
    Figure Legend Snippet: DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the methyltransferase M.CviPI to measure CA in the GpC dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.

    Techniques Used: DNA Methylation Assay, Methylation, Methylation Sequencing, Footprinting, Gel Permeation Chromatography

    2) Product Images from "Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells"

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    Journal: eLife

    doi: 10.7554/eLife.23203

    Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009
    Figure Legend Snippet: Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009

    Techniques Used: Gel Permeation Chromatography, CpG Methylation Assay, Methylation

    Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005
    Figure Legend Snippet: Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005

    Techniques Used: Gel Permeation Chromatography

    Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (
    Figure Legend Snippet: Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (

    Techniques Used: Gel Permeation Chromatography, Methylation

    Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008
    Figure Legend Snippet: Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008

    Techniques Used: Gel Permeation Chromatography, Methylation, CpG Methylation Assay, Activity Assay

    3) Product Images from "Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells"

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    Journal: eLife

    doi: 10.7554/eLife.23203

    Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009
    Figure Legend Snippet: Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009

    Techniques Used: Gel Permeation Chromatography, CpG Methylation Assay, Methylation

    Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005
    Figure Legend Snippet: Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005

    Techniques Used: Gel Permeation Chromatography

    Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (
    Figure Legend Snippet: Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (

    Techniques Used: Gel Permeation Chromatography, Methylation

    Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008
    Figure Legend Snippet: Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008

    Techniques Used: Gel Permeation Chromatography, Methylation, CpG Methylation Assay, Activity Assay

    4) Product Images from "Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing"

    Article Title: Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

    Journal: bioRxiv

    doi: 10.1101/504993

    NanoNOMe can measure endogenous DNA methylation and accessibility. ( a ) nanoNOMe uses exogenous methylation of accessible DNA by GpC methyltransferase, followed by nanopore sequencing and nanopolish methylation detection. ( b ) Using bisulfite mode of IGV to visualize methylation on nanopore sequencing reads versus bisulfite sequencing. Note gaps in coverage in highly repetitive region. ( c ) Aggregate plots of endogenous methylation (left) and chromatin accessibility (right) from nanoNOMe (red) and conventional assays (BS-seq and MNase-seq, blue) centered around CTCF binding sites.
    Figure Legend Snippet: NanoNOMe can measure endogenous DNA methylation and accessibility. ( a ) nanoNOMe uses exogenous methylation of accessible DNA by GpC methyltransferase, followed by nanopore sequencing and nanopolish methylation detection. ( b ) Using bisulfite mode of IGV to visualize methylation on nanopore sequencing reads versus bisulfite sequencing. Note gaps in coverage in highly repetitive region. ( c ) Aggregate plots of endogenous methylation (left) and chromatin accessibility (right) from nanoNOMe (red) and conventional assays (BS-seq and MNase-seq, blue) centered around CTCF binding sites.

    Techniques Used: DNA Methylation Assay, Methylation, Gel Permeation Chromatography, Nanopore Sequencing, Methylation Sequencing, Binding Assay

    5) Product Images from "Single nucleus multi-omics links human cortical cell regulatory genome diversity to disease risk variants"

    Article Title: Single nucleus multi-omics links human cortical cell regulatory genome diversity to disease risk variants

    Journal: bioRxiv

    doi: 10.1101/2019.12.11.873398

    snmC2T-seq generates single-nucleus multi-omic profiles from human brain tissues. (A) The fraction of total snmC2T-seq reads derived from methylome and transcriptome. (B) The fraction of snmC2T-seq transcriptome reads mapped to exons, introns or gene bodies. (C) Boxplot comparing the number of reads detected in each cell/nucleus by different single-cell or single-nucleus RNA-seq technologies. (D-G) snmC2T-seq methylome was compared to other single-cell methylome methods with respect to mapping rate (D), library complexity (E), enrichment of CpG islands (F) and coverage uniformity (G). (H-J) UMAP embedding of 4253 snmC2T-seq cells using single modality information: transcriptome (H), methylome (mCH and mCG, I) and chromatin accessibility (J). (K-L) Pearson correlation of gene expression levels quantified by snmC2T-seq transcriptome and snRNA-seq in MGE PVALB (K) and L1-3 CUX2 (L) cells. (M) Pearson correlation of gene body non-CG methylation quantified with snmC2T-seq methylome and snmC-seq for MGE PVALB cells. (N) Pearson correlation of CG methylation at DMRs quantified with snmC2T-seq methylome and snmC-seq for MGE PVALB cells. (O) Genome-wide methylation level for all tri-nucleotide context (−1 to +2 position) surrounding cytosines shows the sequence specificity of GpC methyltransferase M.CviPI. (P-Q) Spearman correlation between the frequency of methylated GCY sites and ATAC-seq signal at open chromatin sites in L1-3 CUX2 (P) and Oligodendrocyte (Q) cells. (R-S) Overlap of open chromatin peaks identified by snmC2T-seq and snATAC-seq in L1-3 CUX2 (R) and oligodendrocyte (S) cells.
    Figure Legend Snippet: snmC2T-seq generates single-nucleus multi-omic profiles from human brain tissues. (A) The fraction of total snmC2T-seq reads derived from methylome and transcriptome. (B) The fraction of snmC2T-seq transcriptome reads mapped to exons, introns or gene bodies. (C) Boxplot comparing the number of reads detected in each cell/nucleus by different single-cell or single-nucleus RNA-seq technologies. (D-G) snmC2T-seq methylome was compared to other single-cell methylome methods with respect to mapping rate (D), library complexity (E), enrichment of CpG islands (F) and coverage uniformity (G). (H-J) UMAP embedding of 4253 snmC2T-seq cells using single modality information: transcriptome (H), methylome (mCH and mCG, I) and chromatin accessibility (J). (K-L) Pearson correlation of gene expression levels quantified by snmC2T-seq transcriptome and snRNA-seq in MGE PVALB (K) and L1-3 CUX2 (L) cells. (M) Pearson correlation of gene body non-CG methylation quantified with snmC2T-seq methylome and snmC-seq for MGE PVALB cells. (N) Pearson correlation of CG methylation at DMRs quantified with snmC2T-seq methylome and snmC-seq for MGE PVALB cells. (O) Genome-wide methylation level for all tri-nucleotide context (−1 to +2 position) surrounding cytosines shows the sequence specificity of GpC methyltransferase M.CviPI. (P-Q) Spearman correlation between the frequency of methylated GCY sites and ATAC-seq signal at open chromatin sites in L1-3 CUX2 (P) and Oligodendrocyte (Q) cells. (R-S) Overlap of open chromatin peaks identified by snmC2T-seq and snATAC-seq in L1-3 CUX2 (R) and oligodendrocyte (S) cells.

    Techniques Used: Derivative Assay, RNA Sequencing Assay, Expressing, Methylation, Genome Wide, Sequencing, Gel Permeation Chromatography

    6) Product Images from "Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells"

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    Journal: bioRxiv

    doi: 10.1101/061739

    scNOMe-seq detected DNase hypersensitive sites in single cells. a) Schematic of GpC methyltransferase-based mapping of chromatin accessibility and simultaneous detection of endogenous DNA methylation. b) Schematic of scNOMe-seq procedure introduced in this study.
    Figure Legend Snippet: scNOMe-seq detected DNase hypersensitive sites in single cells. a) Schematic of GpC methyltransferase-based mapping of chromatin accessibility and simultaneous detection of endogenous DNA methylation. b) Schematic of scNOMe-seq procedure introduced in this study.

    Techniques Used: Gel Permeation Chromatography, DNA Methylation Assay

    7) Product Images from "GTAGMe-seq: joint profiling of genetic variants, DNA methylation, GpC methyltransferase footprints, and 3D genome in the same DNA molecules"

    Article Title: GTAGMe-seq: joint profiling of genetic variants, DNA methylation, GpC methyltransferase footprints, and 3D genome in the same DNA molecules

    Journal: bioRxiv

    doi: 10.1101/2022.03.29.486102

    GTAGMe-seq generates high-quality multi-omics data. (a). The method scheme. (b). The 3D genome concordance with Hi-C in IMR-90. From left to right: 1Mb resolution across all chromosomes, 250kb resolution for chromosome 6 (include a comparison of compartment score with Hi-C), 50kb resolution for chromosome 6 (include a comparison of insulation score with Hi-C). (c). The methylation concordance with WGBS at HCG (H=A, C, or T) sites in IMR-90. (d). Average GCH (H=A, C, or T) methyltransferase footprint level (left) and HCG methylation level (right) near PolII binding sites. PolII binding sites are divided into quantiles based on the signal strengths of PolII ChIP-seq in IMR-90. Matched random intervals are randomly chosen from the same chromosome and interval lengths as the intervals in top 0-25% quantile. (e). Average GCH methyltransferase footprint level and HCG methylation level around the distal CEBPB binding sites in IMR-90. (f). Scatterplot of the concordance at the gene level with total RNA-seq (ENCODE) in IMR-90. (g). The SNPs concordance with the ground truth SNPs data from Genome in a Bottle (GIAB). The results are compared with the SNPs called from WGS (1000 genome project, phase3 in NA12878). Ti/Tv ratio is also compared.
    Figure Legend Snippet: GTAGMe-seq generates high-quality multi-omics data. (a). The method scheme. (b). The 3D genome concordance with Hi-C in IMR-90. From left to right: 1Mb resolution across all chromosomes, 250kb resolution for chromosome 6 (include a comparison of compartment score with Hi-C), 50kb resolution for chromosome 6 (include a comparison of insulation score with Hi-C). (c). The methylation concordance with WGBS at HCG (H=A, C, or T) sites in IMR-90. (d). Average GCH (H=A, C, or T) methyltransferase footprint level (left) and HCG methylation level (right) near PolII binding sites. PolII binding sites are divided into quantiles based on the signal strengths of PolII ChIP-seq in IMR-90. Matched random intervals are randomly chosen from the same chromosome and interval lengths as the intervals in top 0-25% quantile. (e). Average GCH methyltransferase footprint level and HCG methylation level around the distal CEBPB binding sites in IMR-90. (f). Scatterplot of the concordance at the gene level with total RNA-seq (ENCODE) in IMR-90. (g). The SNPs concordance with the ground truth SNPs data from Genome in a Bottle (GIAB). The results are compared with the SNPs called from WGS (1000 genome project, phase3 in NA12878). Ti/Tv ratio is also compared.

    Techniques Used: Hi-C, Methylation, Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay

    GTAGMe-seq reveals the long-range allele-specific GCH methyltransferase footprint. (a). Scheme and the heatmap representations of the three groups’ long-range GCH methyltransferase footprint. The distance between SNP anchors and non-SNP anchors should be more than 1kb. Group 1 (purple) is the group of loci with significant differences of GCH methyltransferase footprint between Allele A and Allele B at both SNP anchors (FDR
    Figure Legend Snippet: GTAGMe-seq reveals the long-range allele-specific GCH methyltransferase footprint. (a). Scheme and the heatmap representations of the three groups’ long-range GCH methyltransferase footprint. The distance between SNP anchors and non-SNP anchors should be more than 1kb. Group 1 (purple) is the group of loci with significant differences of GCH methyltransferase footprint between Allele A and Allele B at both SNP anchors (FDR

    Techniques Used:

    GTAGMe-seq reveals the coordinated GCH methyltransferase footprint at distal regions in spatial proximity. (a). The coordinated GCH methyltransferase footprint is much stronger at the read pairs from the same DNA molecules (top panel) than that from different DNA molecules but in the same regions (bottom panel). Each dot at the heatmap represents one read pair within the HiCCUPS anchor regions. (b). Average GCH methyltransferase concordance level across all the HiCCUPS anchor regions ( > 20kb, orange color), their matched local regions (utilize the read pairs from the same anchor and within 1kb distance, black color), or matched shuffled read pairs from the same HiCCUPS anchor regions ( > 20kb, blue color).
    Figure Legend Snippet: GTAGMe-seq reveals the coordinated GCH methyltransferase footprint at distal regions in spatial proximity. (a). The coordinated GCH methyltransferase footprint is much stronger at the read pairs from the same DNA molecules (top panel) than that from different DNA molecules but in the same regions (bottom panel). Each dot at the heatmap represents one read pair within the HiCCUPS anchor regions. (b). Average GCH methyltransferase concordance level across all the HiCCUPS anchor regions ( > 20kb, orange color), their matched local regions (utilize the read pairs from the same anchor and within 1kb distance, black color), or matched shuffled read pairs from the same HiCCUPS anchor regions ( > 20kb, blue color).

    Techniques Used:

    8) Product Images from "GTAGMe-seq: joint profiling of genetic variants, DNA methylation, GpC methyltransferase footprints, and 3D genome in the same DNA molecules"

    Article Title: GTAGMe-seq: joint profiling of genetic variants, DNA methylation, GpC methyltransferase footprints, and 3D genome in the same DNA molecules

    Journal: bioRxiv

    doi: 10.1101/2022.03.29.486102

    GTAGMe-seq generates high-quality multi-omics data. (a). The method scheme. (b). The 3D genome concordance with Hi-C in IMR-90. From left to right: 1Mb resolution across all chromosomes, 250kb resolution for chromosome 6 (include a comparison of compartment score with Hi-C), 50kb resolution for chromosome 6 (include a comparison of insulation score with Hi-C). (c). The methylation concordance with WGBS at HCG (H=A, C, or T) sites in IMR-90. (d). Average GCH (H=A, C, or T) methyltransferase footprint level (left) and HCG methylation level (right) near PolII binding sites. PolII binding sites are divided into quantiles based on the signal strengths of PolII ChIP-seq in IMR-90. Matched random intervals are randomly chosen from the same chromosome and interval lengths as the intervals in top 0-25% quantile. (e). Average GCH methyltransferase footprint level and HCG methylation level around the distal CEBPB binding sites in IMR-90. (f). Scatterplot of the concordance at the gene level with total RNA-seq (ENCODE) in IMR-90. (g). The SNPs concordance with the ground truth SNPs data from Genome in a Bottle (GIAB). The results are compared with the SNPs called from WGS (1000 genome project, phase3 in NA12878). Ti/Tv ratio is also compared.
    Figure Legend Snippet: GTAGMe-seq generates high-quality multi-omics data. (a). The method scheme. (b). The 3D genome concordance with Hi-C in IMR-90. From left to right: 1Mb resolution across all chromosomes, 250kb resolution for chromosome 6 (include a comparison of compartment score with Hi-C), 50kb resolution for chromosome 6 (include a comparison of insulation score with Hi-C). (c). The methylation concordance with WGBS at HCG (H=A, C, or T) sites in IMR-90. (d). Average GCH (H=A, C, or T) methyltransferase footprint level (left) and HCG methylation level (right) near PolII binding sites. PolII binding sites are divided into quantiles based on the signal strengths of PolII ChIP-seq in IMR-90. Matched random intervals are randomly chosen from the same chromosome and interval lengths as the intervals in top 0-25% quantile. (e). Average GCH methyltransferase footprint level and HCG methylation level around the distal CEBPB binding sites in IMR-90. (f). Scatterplot of the concordance at the gene level with total RNA-seq (ENCODE) in IMR-90. (g). The SNPs concordance with the ground truth SNPs data from Genome in a Bottle (GIAB). The results are compared with the SNPs called from WGS (1000 genome project, phase3 in NA12878). Ti/Tv ratio is also compared.

    Techniques Used: Hi-C, Methylation, Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay

    GTAGMe-seq reveals the long-range allele-specific GCH methyltransferase footprint. (a). Scheme and the heatmap representations of the three groups’ long-range GCH methyltransferase footprint. The distance between SNP anchors and non-SNP anchors should be more than 1kb. Group 1 (purple) is the group of loci with significant differences of GCH methyltransferase footprint between Allele A and Allele B at both SNP anchors (FDR
    Figure Legend Snippet: GTAGMe-seq reveals the long-range allele-specific GCH methyltransferase footprint. (a). Scheme and the heatmap representations of the three groups’ long-range GCH methyltransferase footprint. The distance between SNP anchors and non-SNP anchors should be more than 1kb. Group 1 (purple) is the group of loci with significant differences of GCH methyltransferase footprint between Allele A and Allele B at both SNP anchors (FDR

    Techniques Used:

    GTAGMe-seq reveals the coordinated GCH methyltransferase footprint at distal regions in spatial proximity. (a). The coordinated GCH methyltransferase footprint is much stronger at the read pairs from the same DNA molecules (top panel) than that from different DNA molecules but in the same regions (bottom panel). Each dot at the heatmap represents one read pair within the HiCCUPS anchor regions. (b). Average GCH methyltransferase concordance level across all the HiCCUPS anchor regions ( > 20kb, orange color), their matched local regions (utilize the read pairs from the same anchor and within 1kb distance, black color), or matched shuffled read pairs from the same HiCCUPS anchor regions ( > 20kb, blue color).
    Figure Legend Snippet: GTAGMe-seq reveals the coordinated GCH methyltransferase footprint at distal regions in spatial proximity. (a). The coordinated GCH methyltransferase footprint is much stronger at the read pairs from the same DNA molecules (top panel) than that from different DNA molecules but in the same regions (bottom panel). Each dot at the heatmap represents one read pair within the HiCCUPS anchor regions. (b). Average GCH methyltransferase concordance level across all the HiCCUPS anchor regions ( > 20kb, orange color), their matched local regions (utilize the read pairs from the same anchor and within 1kb distance, black color), or matched shuffled read pairs from the same HiCCUPS anchor regions ( > 20kb, blue color).

    Techniques Used:

    9) Product Images from "Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation"

    Article Title: Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation

    Journal: bioRxiv

    doi: 10.1101/2022.05.19.492653

    (A) Nucleotide contexts methylated by M.CviPI in vitro . Barplot representing the average methylation levels of lambda DNA in various trinucleotide contexts upon in vitro methylation with M.CviPI in saturating conditions. M.CviPI fully methylates GpCs regardless of the nucleotide context (green bars). It methylates CpGs in GCmG contexts and displays a low level of non-specific activity in CCmG context (
    Figure Legend Snippet: (A) Nucleotide contexts methylated by M.CviPI in vitro . Barplot representing the average methylation levels of lambda DNA in various trinucleotide contexts upon in vitro methylation with M.CviPI in saturating conditions. M.CviPI fully methylates GpCs regardless of the nucleotide context (green bars). It methylates CpGs in GCmG contexts and displays a low level of non-specific activity in CCmG context (

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

    DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the methyltransferase M.CviPI to measure CA in the GpC dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.
    Figure Legend Snippet: DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the methyltransferase M.CviPI to measure CA in the GpC dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.

    Techniques Used: DNA Methylation Assay, Methylation, Methylation Sequencing, Footprinting, Gel Permeation Chromatography

    10) Product Images from "Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression"

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression

    Journal: Human Mutation

    doi: 10.1002/humu.22785

    The variant allele is not epigenetically altered at the MLH1 promoter. A: A schematic of the MLH1 and EPM2AIP1 bidirectional promoter indicating the location of the CpG island (green bar), seven HhaI sites used to detect methylation by MS-MLPA (red vertical bars), and the sequence encompassing the c.-7C > T site. The presence of the c.-7C > T variant abolishes a HhaI restriction site. B: Single molecule bisulfite sequencing data of various tissues from Proband 32. The c.-93G > A site was used to distinguish between wild-type (c.-93G) and variant (c.-93A) MLH1 alleles. The black horizontal bar labeled “Bisulfite seq” indicates the region analyzed. Both alleles were unmethylated in all tissues examined including tumor tissue. C and D: Representative pyrograms indicating methylation levels at five CpG sites within the MLH1 promoter in Proband 32 PBMCs and Proband N buccal DNA, respectively. The nominal limit of quantification for this assay is 5%. E: The locations of unique transcription initiation sites in exon 1a of the wild-type (green box) and variant (blue box) MLH1 alleles. The c.-93, c.-28, and c.-7 sites are indicated by the red vertical bars. F: Nucleosome occupancy across individual promoter molecules separated according to allele of origin, as determined by the c.-7C > T variant (yellow diamond). Black arrows indicate the annotated MLH1 [NM_000249.3] or EPM2AIP1 [NM_014805.3] transcription initiation sites, whereas green and blue arrows indicate the locations of sites identified by 5′RACE in wild-type and variant alleles, respectively. Thin vertical black lines represent the positions of GpC dinucleotides. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M.CviPI. White circles = GpC dinucleotides unmethylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of accessibility ≥150 or > 75 bp at the extreme ends of amplicons. G: Nucleosome occupancy across the same region in MLH1 -expressing colorectal carcinoma cells and PBMCs from healthy donors. The number of molecules sequenced is indicated at the bottom right of each panel.
    Figure Legend Snippet: The variant allele is not epigenetically altered at the MLH1 promoter. A: A schematic of the MLH1 and EPM2AIP1 bidirectional promoter indicating the location of the CpG island (green bar), seven HhaI sites used to detect methylation by MS-MLPA (red vertical bars), and the sequence encompassing the c.-7C > T site. The presence of the c.-7C > T variant abolishes a HhaI restriction site. B: Single molecule bisulfite sequencing data of various tissues from Proband 32. The c.-93G > A site was used to distinguish between wild-type (c.-93G) and variant (c.-93A) MLH1 alleles. The black horizontal bar labeled “Bisulfite seq” indicates the region analyzed. Both alleles were unmethylated in all tissues examined including tumor tissue. C and D: Representative pyrograms indicating methylation levels at five CpG sites within the MLH1 promoter in Proband 32 PBMCs and Proband N buccal DNA, respectively. The nominal limit of quantification for this assay is 5%. E: The locations of unique transcription initiation sites in exon 1a of the wild-type (green box) and variant (blue box) MLH1 alleles. The c.-93, c.-28, and c.-7 sites are indicated by the red vertical bars. F: Nucleosome occupancy across individual promoter molecules separated according to allele of origin, as determined by the c.-7C > T variant (yellow diamond). Black arrows indicate the annotated MLH1 [NM_000249.3] or EPM2AIP1 [NM_014805.3] transcription initiation sites, whereas green and blue arrows indicate the locations of sites identified by 5′RACE in wild-type and variant alleles, respectively. Thin vertical black lines represent the positions of GpC dinucleotides. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M.CviPI. White circles = GpC dinucleotides unmethylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of accessibility ≥150 or > 75 bp at the extreme ends of amplicons. G: Nucleosome occupancy across the same region in MLH1 -expressing colorectal carcinoma cells and PBMCs from healthy donors. The number of molecules sequenced is indicated at the bottom right of each panel.

    Techniques Used: Variant Assay, Methylation, Mass Spectrometry, Multiplex Ligation-dependent Probe Amplification, Sequencing, Methylation Sequencing, Labeling, Gel Permeation Chromatography, Expressing

    11) Product Images from "Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells"

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    Journal: bioRxiv

    doi: 10.1101/061739

    scNOMe-seq detected DNase hypersensitive sites in single cells. a) Schematic of GpC methyltransferase-based mapping of chromatin accessibility and simultaneous detection of endogenous DNA methylation. b) Schematic of scNOMe-seq procedure introduced in this study.
    Figure Legend Snippet: scNOMe-seq detected DNase hypersensitive sites in single cells. a) Schematic of GpC methyltransferase-based mapping of chromatin accessibility and simultaneous detection of endogenous DNA methylation. b) Schematic of scNOMe-seq procedure introduced in this study.

    Techniques Used: Gel Permeation Chromatography, DNA Methylation Assay

    12) Product Images from "Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation"

    Article Title: Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation

    Journal: bioRxiv

    doi: 10.1101/2022.05.19.492653

    (A) Nucleotide contexts methylated by M.CviPI in vitro . Barplot representing the average methylation levels of lambda DNA in various trinucleotide contexts upon in vitro methylation with M.CviPI in saturating conditions. M.CviPI fully methylates GpCs regardless of the nucleotide context (green bars). It methylates CpGs in GCmG contexts and displays a low level of non-specific activity in CCmG context (
    Figure Legend Snippet: (A) Nucleotide contexts methylated by M.CviPI in vitro . Barplot representing the average methylation levels of lambda DNA in various trinucleotide contexts upon in vitro methylation with M.CviPI in saturating conditions. M.CviPI fully methylates GpCs regardless of the nucleotide context (green bars). It methylates CpGs in GCmG contexts and displays a low level of non-specific activity in CCmG context (

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

    DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the methyltransferase M.CviPI to measure CA in the GpC dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.
    Figure Legend Snippet: DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the methyltransferase M.CviPI to measure CA in the GpC dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.

    Techniques Used: DNA Methylation Assay, Methylation, Methylation Sequencing, Footprinting, Gel Permeation Chromatography

    13) Product Images from "Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression"

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression

    Journal: Human Mutation

    doi: 10.1002/humu.22785

    The variant allele is not epigenetically altered at the MLH1 promoter. A: A schematic of the MLH1 and EPM2AIP1 bidirectional promoter indicating the location of the CpG island (green bar), seven HhaI sites used to detect methylation by MS-MLPA (red vertical bars), and the sequence encompassing the c.-7C > T site. The presence of the c.-7C > T variant abolishes a HhaI restriction site. B: Single molecule bisulfite sequencing data of various tissues from Proband 32. The c.-93G > A site was used to distinguish between wild-type (c.-93G) and variant (c.-93A) MLH1 alleles. The black horizontal bar labeled “Bisulfite seq” indicates the region analyzed. Both alleles were unmethylated in all tissues examined including tumor tissue. C and D: Representative pyrograms indicating methylation levels at five CpG sites within the MLH1 promoter in Proband 32 PBMCs and Proband N buccal DNA, respectively. The nominal limit of quantification for this assay is 5%. E: The locations of unique transcription initiation sites in exon 1a of the wild-type (green box) and variant (blue box) MLH1 alleles. The c.-93, c.-28, and c.-7 sites are indicated by the red vertical bars. F: Nucleosome occupancy across individual promoter molecules separated according to allele of origin, as determined by the c.-7C > T variant (yellow diamond). Black arrows indicate the annotated MLH1 [NM_000249.3] or EPM2AIP1 [NM_014805.3] transcription initiation sites, whereas green and blue arrows indicate the locations of sites identified by 5′RACE in wild-type and variant alleles, respectively. Thin vertical black lines represent the positions of GpC dinucleotides. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M.CviPI. White circles = GpC dinucleotides unmethylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of accessibility ≥150 or > 75 bp at the extreme ends of amplicons. G: Nucleosome occupancy across the same region in MLH1 -expressing colorectal carcinoma cells and PBMCs from healthy donors. The number of molecules sequenced is indicated at the bottom right of each panel.
    Figure Legend Snippet: The variant allele is not epigenetically altered at the MLH1 promoter. A: A schematic of the MLH1 and EPM2AIP1 bidirectional promoter indicating the location of the CpG island (green bar), seven HhaI sites used to detect methylation by MS-MLPA (red vertical bars), and the sequence encompassing the c.-7C > T site. The presence of the c.-7C > T variant abolishes a HhaI restriction site. B: Single molecule bisulfite sequencing data of various tissues from Proband 32. The c.-93G > A site was used to distinguish between wild-type (c.-93G) and variant (c.-93A) MLH1 alleles. The black horizontal bar labeled “Bisulfite seq” indicates the region analyzed. Both alleles were unmethylated in all tissues examined including tumor tissue. C and D: Representative pyrograms indicating methylation levels at five CpG sites within the MLH1 promoter in Proband 32 PBMCs and Proband N buccal DNA, respectively. The nominal limit of quantification for this assay is 5%. E: The locations of unique transcription initiation sites in exon 1a of the wild-type (green box) and variant (blue box) MLH1 alleles. The c.-93, c.-28, and c.-7 sites are indicated by the red vertical bars. F: Nucleosome occupancy across individual promoter molecules separated according to allele of origin, as determined by the c.-7C > T variant (yellow diamond). Black arrows indicate the annotated MLH1 [NM_000249.3] or EPM2AIP1 [NM_014805.3] transcription initiation sites, whereas green and blue arrows indicate the locations of sites identified by 5′RACE in wild-type and variant alleles, respectively. Thin vertical black lines represent the positions of GpC dinucleotides. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M.CviPI. White circles = GpC dinucleotides unmethylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of accessibility ≥150 or > 75 bp at the extreme ends of amplicons. G: Nucleosome occupancy across the same region in MLH1 -expressing colorectal carcinoma cells and PBMCs from healthy donors. The number of molecules sequenced is indicated at the bottom right of each panel.

    Techniques Used: Variant Assay, Methylation, Mass Spectrometry, Multiplex Ligation-dependent Probe Amplification, Sequencing, Methylation Sequencing, Labeling, Gel Permeation Chromatography, Expressing

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    New England Biolabs gpc methyltransferase treatment
    DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the <t>methyltransferase</t> M.CviPI to measure CA in the <t>GpC</t> dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.
    Gpc Methyltransferase Treatment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the methyltransferase M.CviPI to measure CA in the GpC dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.

    Journal: bioRxiv

    Article Title: Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation

    doi: 10.1101/2022.05.19.492653

    Figure Lengend Snippet: DNA methylation is neutral to chromatin accessibility at the majority of cis -regulatory elements. (A) Single locus example of the heterogeneity in DNA methylation (5mC) levels at enhancers and promoters when resolved on individual molecules. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by Whole Genome Bisulfite Sequencing (WGBS) around the Macf1 promoter and its intragenic enhancer. Lower panel: At the molecular level, 5mC at enhancers is heterogeneous. 5mC of individual CpGs (red) is shown at molecular level (blue methylated, white unmethylated). (B) Schematic representation of the experimental strategy used to identify molecular antagonisms between 5mC and chromatin accessibility (CA). Single molecule footprinting (SMF) is performed using the methyltransferase M.CviPI to measure CA in the GpC dinucleotide context, that is distinct from the endogenous 5mC in CpG context. Bisulfite sequencing of the DNA provides continuous information on CA and 5mC over 300 bp long DNA molecules. The accessibility of each molecule is calculated using the methylation of GpCs within the 100 bp surrounding a CpG. The average accessibility is used to classify DNA molecules into accessible and inaccessible fractions. In both fractions, the average 5mC levels are calculated. White/blue lollipops represent the endogenous un-/methylated state, white/black lollipops represent the CA as measured by SMF. (C) Most intermediately methylated CpGs show no difference in 5mC between the two separated CA fractions. Violin bar plots showing the CA (top) and 5mC (bottom) at CpGs in bulk and in the two separated fractions (n = 97,808). Boxplots show median (black middle line), 25th and 75th percentiles (black boundaries). (D) Single locus example of the 5mC and CA of individual molecules at a neutral CpG. Upper panel: Genome browser track displaying the chromatin marks and the average methylation as measured by WGBS around an intergenic enhancer. Middle panel: Depicted is the average SMF signal (1 methylation %) of individual GpCs (black dots) and endogenous 5mC of individual CpGs (blue diamonds). Lower panels: Depicted are the single DNA molecules mapped to this genomic locus sorted by CA into an accessible (grey) and an inaccessible (black) fraction, and the 5mC of the molecules in both fractions. In the lower left panel, every column is a single GpC dinucleotide depicting its accessibility status (grey: accessible, black: inaccessible). In the lower right panel, every column is a single CpG dinucleotide depicting its methylation status (light blue: unmethylated, dark blue: methylated). Percentages represent average 5mC of the CpG of interest at the center in each fraction. The number of single DNA molecules used for the analysis is indicated.

    Article Snippet: For the GpC methyltransferase treatment, freshly made GpC methyltransferase mix (1x M.GpC buffer, 300 mM sucrose, 64 µM SAM (NEB, #B9003S)) and 200 U M.CviPI (NEB, #M0227L) were added and incubated at 37°C for 7.5 min. For a second incubation round at 37°C for 7.5 min the reaction was replenished with 100U of M.CviPI and 128 pmol of SAM.

    Techniques: DNA Methylation Assay, Methylation, Methylation Sequencing, Footprinting, Gel Permeation Chromatography

    Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009

    Journal: eLife

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    doi: 10.7554/eLife.23203

    Figure Lengend Snippet: Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009

    Article Snippet: One million nuclei were resupended in reaction buffer to yield a suspension with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB), and 50 ul of GpC methyltransferase (4 U/ul)) from M.CviPI (NEB).

    Techniques: Gel Permeation Chromatography, CpG Methylation Assay, Methylation

    Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005

    Journal: eLife

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    doi: 10.7554/eLife.23203

    Figure Lengend Snippet: Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005

    Article Snippet: One million nuclei were resupended in reaction buffer to yield a suspension with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB), and 50 ul of GpC methyltransferase (4 U/ul)) from M.CviPI (NEB).

    Techniques: Gel Permeation Chromatography

    Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (

    Journal: eLife

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    doi: 10.7554/eLife.23203

    Figure Lengend Snippet: Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (

    Article Snippet: One million nuclei were resupended in reaction buffer to yield a suspension with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB), and 50 ul of GpC methyltransferase (4 U/ul)) from M.CviPI (NEB).

    Techniques: Gel Permeation Chromatography, Methylation

    Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008

    Journal: eLife

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    doi: 10.7554/eLife.23203

    Figure Lengend Snippet: Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008

    Article Snippet: One million nuclei were resupended in reaction buffer to yield a suspension with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB), and 50 ul of GpC methyltransferase (4 U/ul)) from M.CviPI (NEB).

    Techniques: Gel Permeation Chromatography, Methylation, CpG Methylation Assay, Activity Assay

    NanoNOMe can measure endogenous DNA methylation and accessibility. ( a ) nanoNOMe uses exogenous methylation of accessible DNA by GpC methyltransferase, followed by nanopore sequencing and nanopolish methylation detection. ( b ) Using bisulfite mode of IGV to visualize methylation on nanopore sequencing reads versus bisulfite sequencing. Note gaps in coverage in highly repetitive region. ( c ) Aggregate plots of endogenous methylation (left) and chromatin accessibility (right) from nanoNOMe (red) and conventional assays (BS-seq and MNase-seq, blue) centered around CTCF binding sites.

    Journal: bioRxiv

    Article Title: Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

    doi: 10.1101/504993

    Figure Lengend Snippet: NanoNOMe can measure endogenous DNA methylation and accessibility. ( a ) nanoNOMe uses exogenous methylation of accessible DNA by GpC methyltransferase, followed by nanopore sequencing and nanopolish methylation detection. ( b ) Using bisulfite mode of IGV to visualize methylation on nanopore sequencing reads versus bisulfite sequencing. Note gaps in coverage in highly repetitive region. ( c ) Aggregate plots of endogenous methylation (left) and chromatin accessibility (right) from nanoNOMe (red) and conventional assays (BS-seq and MNase-seq, blue) centered around CTCF binding sites.

    Article Snippet: The resulting unmethylated, sheared DNA was methylated with M. SssI (NEB Cat. M0226) for CpG methylation or M. CviPI (NEB Cat. M0227) for GpC methylation, or both enzymes for CpG+GpC methylation.

    Techniques: DNA Methylation Assay, Methylation, Gel Permeation Chromatography, Nanopore Sequencing, Methylation Sequencing, Binding Assay