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    New England Biolabs gpc methyltransferase m cvipi
    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 <t>GpC</t> methyltransferase <t>M.CviPI.</t> (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.
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    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.

    Journal: bioRxiv

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

    doi: 10.1101/2019.12.11.873398

    Figure Lengend 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.

    Article Snippet: One million nuclei aliquots were pelleted by 1,000 x g at 4°C for 10 min and resuspended in 200 µl of GpC methyltransferase M.CviPI (NEB M0227L) reaction containing 1X GC Reaction Buffer, 0.32 nM S-Adenoslylmethionime, 80U 4U/µl M.CviPI, 1:100 SUPERaseIn RNase Inhibitor and 1:100 RNaseOUT RNase Inhibitor and incubated at 37°C for 8 min.

    Techniques: Derivative Assay, RNA Sequencing Assay, Expressing, Methylation, Genome Wide, Sequencing, 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

    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.

    Journal: Human Mutation

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

    doi: 10.1002/humu.22785

    Figure Lengend 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.

    Article Snippet: The promoter of the HSPA5 (MIM #138120) gene, known to be nucleosome free and accessible, was used as a control for GpC methyltransferase M.CviPl in each sample examined.

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