gpc methyltransferase treatment  (New England Biolabs)

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

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

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

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

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

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