gpc methyltransferase m cvipl  (New England Biolabs)


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    Name:
    GpC Methyltransferase M CviPI
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    GpC Methyltransferase M CviPI 1 000 units
    Catalog Number:
    m0227l
    Price:
    308
    Size:
    1 000 units
    Category:
    DNA Methylases
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    New England Biolabs gpc methyltransferase m cvipl
    GpC Methyltransferase M CviPI
    GpC Methyltransferase M CviPI 1 000 units
    https://www.bioz.com/result/gpc methyltransferase m cvipl/product/New England Biolabs
    Average 95 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    gpc methyltransferase m cvipl - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) 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

    2) 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

    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

    Related Articles

    Clone Assay:

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression
    Article Snippet: This involved harvesting intact nuclei and treating with 200 U GpC methyltransferase M.CviPl (New England Biolabs, Ipswich, MA) for 15 min at 37°C followed by termination of the reaction with an equal volume of 20 mM Tris HCl pH 7.9, 600 mM NaCl, 1% (w/v) SDS, and 10 mM EDTA and overnight digestion with 200 μg/ml Proteinase K (Ambion, Austin, TX). .. PCR amplicons were cloned using the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA) and individual molecules isolated by colony PCR for sequencing as described previously [Hesson and Ward, ].

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: Intact nuclei were treated with 200 U of the GpC methyltransferase M.CviPI (New England BioLabs) and SAM for 8 min at 37°, followed by an additional 100 U of M.CviPI and SAM for 8 min at 37°. .. Regions of DUX4 myogenic enhancer 1 (DME1), DME2, and the MyoD core enhancer were PCR amplified using primers devoid of GpC or CpG dinucleotides and then TA cloned and sequenced to determine patterns of CpG and GpC methylation.

    Centrifugation:

    Article Title: Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum
    Article Snippet: Taq DNA polymerase, restriction endonucleases, CpG (M.SssI) and GpC (M.CviPI) methyltransferases, Quick Ligation Kit, and 1 kb DNA ladder were purchased from NEB (Ipswich, MA, USA). .. Briefly, 3–9 ml of late-exponential phase cells were collected by centrifugation and washed twice in KET buffer (0.5 M KCl, 0.1 M EDTA, and 0.05 M Tris–HCl, pH 8.0) and once in SET buffer (25% sucrose, 0.05 M EDTA, and 0.05 M Tris–HCl, pH 8.0).

    Amplification:

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C. .. Briefly, the isolated genomic DNA was first bisulfite converted using MethylCode Bisulfite Conversion Kit (ThermoFisher #MECOV-50), and first strand was synthesized using random nonamer primers with biotin-tagged truncated Illumina P5 adapter (5′-biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′), and second strands were synthesized using random nonamer primers containing a truncated P7 Illumina adapter (5′-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3′), the final library was amplified with 4-6 cycles of PCR using 1 U Kapa HiFi HS DNA Polymerase (KAPA Biosystems), and then AMpure XP Beads (Beckman Coulter) purified, quantified and pooled on Illumina HiSeq 2500 platform with 100 or 150 bp paired-end mode (sequenced by Novogene).

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: Next, the bisulfite-treated genomic DNA from a single cell was amplified by a PBAT strategy to obtain sufficient material for sequencing. .. Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM.

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: Intact nuclei were treated with 200 U of the GpC methyltransferase M.CviPI (New England BioLabs) and SAM for 8 min at 37°, followed by an additional 100 U of M.CviPI and SAM for 8 min at 37°. .. Regions of DUX4 myogenic enhancer 1 (DME1), DME2, and the MyoD core enhancer were PCR amplified using primers devoid of GpC or CpG dinucleotides and then TA cloned and sequenced to determine patterns of CpG and GpC methylation.

    Synthesized:

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C. .. Briefly, the isolated genomic DNA was first bisulfite converted using MethylCode Bisulfite Conversion Kit (ThermoFisher #MECOV-50), and first strand was synthesized using random nonamer primers with biotin-tagged truncated Illumina P5 adapter (5′-biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′), and second strands were synthesized using random nonamer primers containing a truncated P7 Illumina adapter (5′-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3′), the final library was amplified with 4-6 cycles of PCR using 1 U Kapa HiFi HS DNA Polymerase (KAPA Biosystems), and then AMpure XP Beads (Beckman Coulter) purified, quantified and pooled on Illumina HiSeq 2500 platform with 100 or 150 bp paired-end mode (sequenced by Novogene).

    Lambda DNA Preparation:

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: .. The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C. ..

    TA Cloning:

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression
    Article Snippet: This involved harvesting intact nuclei and treating with 200 U GpC methyltransferase M.CviPl (New England Biolabs, Ipswich, MA) for 15 min at 37°C followed by termination of the reaction with an equal volume of 20 mM Tris HCl pH 7.9, 600 mM NaCl, 1% (w/v) SDS, and 10 mM EDTA and overnight digestion with 200 μg/ml Proteinase K (Ambion, Austin, TX). .. PCR amplicons were cloned using the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA) and individual molecules isolated by colony PCR for sequencing as described previously [Hesson and Ward, ].

    Enzyme-linked Immunosorbent Assay:

    Article Title: Age-associated epigenetic modifications in human DNA increase its immunogenicity
    Article Snippet: In this kit the methylated fraction of DNA is recognized by 5-methylcytosine antibody and quantified through an ELISA-like reaction. .. 1μg of DNA was methylated in GC Reaction Buffer containing 60 units of GpC Methyltransferase (M.CviPI) and 160 μM S-adenosylmethionine from New England Biolabs (Ipswich, MA) at 37o C for either 4 or 18 hours.

    Incubation:

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM. .. After in vitro methylation, 0.5 μl of 20 mg/ml protease (Qiagen) was added and the mixture was incubated for 3 h at 50 °C to release genomic DNA.

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells
    Article Snippet: Between 2 × 10^6 and 5 × 10∧ 6 cells were harvested by centrifuging the cell suspension for 5 min at 500x g. Cells were washed once with 1x PBS, re-suspended in 1 ml lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) and incubated for 10 min on ice. .. 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).

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: .. Taking advantage of the fact that mammalian cells lack GpC methylation, intact nuclei are incubated with the GpC methyltransferase M.CviPI, which methylates GpC dinucleotides not associated with nucleosomes or tightly bound transcription factors while leaving nucleosomal or transcription factor-occupied sites unmethylated. .. Following bisulfite conversion, patterns of GpC and CpG methylation are assessed, allowing the determination of both nucleosome occupancy and endogenous methylation status for individual DNA strands.

    Modification:

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells
    Article Snippet: NOMe-Seq procedure was performed based on protocols for CpG methyltransferase M.SSsI described in and the GpC methyltransferase from M.CviPI , with some modification. .. 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).

    Ligation:

    Article Title: Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum
    Article Snippet: .. Taq DNA polymerase, restriction endonucleases, CpG (M.SssI) and GpC (M.CviPI) methyltransferases, Quick Ligation Kit, and 1 kb DNA ladder were purchased from NEB (Ipswich, MA, USA). .. Pfu DNA polymerase and RNase A were purchased from Bio Basic, Inc. (Markham, ON, Canada).

    Protease Inhibitor:

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: Briefly, cell pellet was first lysed in 1× lysis buffer (part of the NOMe-Seq Kit, Active motif #54000) with Protease inhibitor and PMSF (part of the NOMe-Seq Kit, Active motif #54000) on ice for 1 h with intermittent vortex. .. The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C.

    Genomic Sequencing:

    Article Title: A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome
    Article Snippet: Chromatin was treated with the M.CviPI methyltransferase (Cat # M0227B; New England Bio Labs, Ipswich, MA), which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin. .. Following bisulfite treatment of the M.CviPI-methylated chromatin and subsequent genomic sequencing, we identified endogenous methylation (CpG) and accessibility (GpC) in the same sequencing reaction.

    Transferring:

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: .. Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM. .. In vitro methylation of single-cell nuclei was performed by incubating the mixture in a thermocycler at 37 °C for 30 min before heat inactivation for 20 min at 65 °C.

    Cell Culture:

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells
    Article Snippet: Paragraph title: Cell culture, nuclei isolation, and GpC methylase treatment ... 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).

    Generated:

    Article Title: A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome
    Article Snippet: Chromatin was treated with the M.CviPI methyltransferase (Cat # M0227B; New England Bio Labs, Ipswich, MA), which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin. .. The RWPE1 NOMe-seq library was sequenced using an Illumina HiSeq 2000 (100 bp paired-end), and NOMe-seq data were deposited in the NCBI GEO accession number, GSE118629; the C42B NOMe-seq library was previously generated (GSE102616).

    Polymerase Chain Reaction:

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression
    Article Snippet: This involved harvesting intact nuclei and treating with 200 U GpC methyltransferase M.CviPl (New England Biolabs, Ipswich, MA) for 15 min at 37°C followed by termination of the reaction with an equal volume of 20 mM Tris HCl pH 7.9, 600 mM NaCl, 1% (w/v) SDS, and 10 mM EDTA and overnight digestion with 200 μg/ml Proteinase K (Ambion, Austin, TX). .. PCR amplicons were cloned using the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA) and individual molecules isolated by colony PCR for sequencing as described previously [Hesson and Ward, ].

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C. .. Briefly, the isolated genomic DNA was first bisulfite converted using MethylCode Bisulfite Conversion Kit (ThermoFisher #MECOV-50), and first strand was synthesized using random nonamer primers with biotin-tagged truncated Illumina P5 adapter (5′-biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′), and second strands were synthesized using random nonamer primers containing a truncated P7 Illumina adapter (5′-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3′), the final library was amplified with 4-6 cycles of PCR using 1 U Kapa HiFi HS DNA Polymerase (KAPA Biosystems), and then AMpure XP Beads (Beckman Coulter) purified, quantified and pooled on Illumina HiSeq 2500 platform with 100 or 150 bp paired-end mode (sequenced by Novogene).

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: .. Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM. .. In vitro methylation of single-cell nuclei was performed by incubating the mixture in a thermocycler at 37 °C for 30 min before heat inactivation for 20 min at 65 °C.

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: Intact nuclei were treated with 200 U of the GpC methyltransferase M.CviPI (New England BioLabs) and SAM for 8 min at 37°, followed by an additional 100 U of M.CviPI and SAM for 8 min at 37°. .. Regions of DUX4 myogenic enhancer 1 (DME1), DME2, and the MyoD core enhancer were PCR amplified using primers devoid of GpC or CpG dinucleotides and then TA cloned and sequenced to determine patterns of CpG and GpC methylation.

    Recombinant:

    Article Title: Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum
    Article Snippet: Recombinant DNA manipulations were performed according to standard procedures [ ]. .. Taq DNA polymerase, restriction endonucleases, CpG (M.SssI) and GpC (M.CviPI) methyltransferases, Quick Ligation Kit, and 1 kb DNA ladder were purchased from NEB (Ipswich, MA, USA).

    DNA Extraction:

    Article Title: Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum
    Article Snippet: Paragraph title: DNA Isolation and manipulation ... Taq DNA polymerase, restriction endonucleases, CpG (M.SssI) and GpC (M.CviPI) methyltransferases, Quick Ligation Kit, and 1 kb DNA ladder were purchased from NEB (Ipswich, MA, USA).

    In Vivo:

    Article Title: Single-molecule long-read sequencing reveals the chromatin basis of gene expression
    Article Snippet: GpC-specific methyltransferase M.CviPI (NEB) supplemented with 160 µM SAM S-adenosylmethionine was used to methylate spheroplasts at 37°C for 45 min. Genomic DNA was extracted using PCI (phenol:chloroform:isoamyl alcohol, 25:24:1) and purified by a Genomic DNA Clean & Concentrator-10 kit (Zymo Research). .. We denote the above mentioned genomic DNA that undergoes in vivo spheroplast methylation as the target sample of MeSMLR-seq.

    Methylation:

    Article Title: A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome
    Article Snippet: Chromatin was treated with the M.CviPI methyltransferase (Cat # M0227B; New England Bio Labs, Ipswich, MA), which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin. .. Following bisulfite treatment of the M.CviPI-methylated chromatin and subsequent genomic sequencing, we identified endogenous methylation (CpG) and accessibility (GpC) in the same sequencing reaction.

    Article Title: Single-cell nucleosome mapping reveals the molecular basis of gene expression heterogeneity
    Article Snippet: Paragraph title: Methylation and Bisulfite Conversion of DNA. ... Spheroplasts were washed with 1 M sorbitol three times before treatment with GpC methyltransferase and treated with 30 units of M.CviP (NEB) supplemented with 160 µM S-adenosylmethionine (NEB) at 37 °C for 45 min.

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression
    Article Snippet: This involved harvesting intact nuclei and treating with 200 U GpC methyltransferase M.CviPl (New England Biolabs, Ipswich, MA) for 15 min at 37°C followed by termination of the reaction with an equal volume of 20 mM Tris HCl pH 7.9, 600 mM NaCl, 1% (w/v) SDS, and 10 mM EDTA and overnight digestion with 200 μg/ml Proteinase K (Ambion, Austin, TX). .. DNA was subsequently isolated and bisulfite converted using EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA).

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: Paragraph title: Preparation and in vitro methylation of single-cell nuclei ... Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM.

    Article Title: Age-associated epigenetic modifications in human DNA increase its immunogenicity
    Article Snippet: .. 1μg of DNA was methylated in GC Reaction Buffer containing 60 units of GpC Methyltransferase (M.CviPI) and 160 μM S-adenosylmethionine from New England Biolabs (Ipswich, MA) at 37o C for either 4 or 18 hours. .. The GC Methyltransferase methylates all cytosine residues within the double stranded recognition sequence of 5'..GC..3'.

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: Intact nuclei were treated with 200 U of the GpC methyltransferase M.CviPI (New England BioLabs) and SAM for 8 min at 37°, followed by an additional 100 U of M.CviPI and SAM for 8 min at 37°. .. Regions of DUX4 myogenic enhancer 1 (DME1), DME2, and the MyoD core enhancer were PCR amplified using primers devoid of GpC or CpG dinucleotides and then TA cloned and sequenced to determine patterns of CpG and GpC methylation.

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: .. Taking advantage of the fact that mammalian cells lack GpC methylation, intact nuclei are incubated with the GpC methyltransferase M.CviPI, which methylates GpC dinucleotides not associated with nucleosomes or tightly bound transcription factors while leaving nucleosomal or transcription factor-occupied sites unmethylated. .. Following bisulfite conversion, patterns of GpC and CpG methylation are assessed, allowing the determination of both nucleosome occupancy and endogenous methylation status for individual DNA strands.

    Article Title: Single-molecule long-read sequencing reveals the chromatin basis of gene expression
    Article Snippet: Preparation and methylation of yeast spheroplasts were performed as previously described ( ; ). .. GpC-specific methyltransferase M.CviPI (NEB) supplemented with 160 µM SAM S-adenosylmethionine was used to methylate spheroplasts at 37°C for 45 min. Genomic DNA was extracted using PCI (phenol:chloroform:isoamyl alcohol, 25:24:1) and purified by a Genomic DNA Clean & Concentrator-10 kit (Zymo Research).

    Isolation:

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression
    Article Snippet: This involved harvesting intact nuclei and treating with 200 U GpC methyltransferase M.CviPl (New England Biolabs, Ipswich, MA) for 15 min at 37°C followed by termination of the reaction with an equal volume of 20 mM Tris HCl pH 7.9, 600 mM NaCl, 1% (w/v) SDS, and 10 mM EDTA and overnight digestion with 200 μg/ml Proteinase K (Ambion, Austin, TX). .. DNA was subsequently isolated and bisulfite converted using EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA).

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C. .. Briefly, the isolated genomic DNA was first bisulfite converted using MethylCode Bisulfite Conversion Kit (ThermoFisher #MECOV-50), and first strand was synthesized using random nonamer primers with biotin-tagged truncated Illumina P5 adapter (5′-biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′), and second strands were synthesized using random nonamer primers containing a truncated P7 Illumina adapter (5′-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3′), the final library was amplified with 4-6 cycles of PCR using 1 U Kapa HiFi HS DNA Polymerase (KAPA Biosystems), and then AMpure XP Beads (Beckman Coulter) purified, quantified and pooled on Illumina HiSeq 2500 platform with 100 or 150 bp paired-end mode (sequenced by Novogene).

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: Preparation and in vitro methylation of single-cell nuclei We first prepared a cell lysate, methylated the chromatin in vitro , released genomic DNA from the chromatin, and treated the genomic DNA with bisulfite in a one-tube reaction to avoid loss of material in the isolation and purification steps. .. Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM.

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells
    Article Snippet: Paragraph title: Cell culture, nuclei isolation, and GpC methylase treatment ... 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).

    Purification:

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C. .. Briefly, the isolated genomic DNA was first bisulfite converted using MethylCode Bisulfite Conversion Kit (ThermoFisher #MECOV-50), and first strand was synthesized using random nonamer primers with biotin-tagged truncated Illumina P5 adapter (5′-biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′), and second strands were synthesized using random nonamer primers containing a truncated P7 Illumina adapter (5′-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3′), the final library was amplified with 4-6 cycles of PCR using 1 U Kapa HiFi HS DNA Polymerase (KAPA Biosystems), and then AMpure XP Beads (Beckman Coulter) purified, quantified and pooled on Illumina HiSeq 2500 platform with 100 or 150 bp paired-end mode (sequenced by Novogene).

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: Preparation and in vitro methylation of single-cell nuclei We first prepared a cell lysate, methylated the chromatin in vitro , released genomic DNA from the chromatin, and treated the genomic DNA with bisulfite in a one-tube reaction to avoid loss of material in the isolation and purification steps. .. Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM.

    Article Title: Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum
    Article Snippet: DNA Isolation and manipulation Plasmid DNA was extracted and purified from E. coli DH5α and ER1821 using an EZ-10 Spin Column Plasmid DNA Miniprep Kit from Bio Basic, Inc. (Markham, ON, Canada). .. Taq DNA polymerase, restriction endonucleases, CpG (M.SssI) and GpC (M.CviPI) methyltransferases, Quick Ligation Kit, and 1 kb DNA ladder were purchased from NEB (Ipswich, MA, USA).

    Article Title: Single-molecule long-read sequencing reveals the chromatin basis of gene expression
    Article Snippet: .. GpC-specific methyltransferase M.CviPI (NEB) supplemented with 160 µM SAM S-adenosylmethionine was used to methylate spheroplasts at 37°C for 45 min. Genomic DNA was extracted using PCI (phenol:chloroform:isoamyl alcohol, 25:24:1) and purified by a Genomic DNA Clean & Concentrator-10 kit (Zymo Research). .. We denote the above mentioned genomic DNA that undergoes in vivo spheroplast methylation as the target sample of MeSMLR-seq.

    Sequencing:

    Article Title: A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome
    Article Snippet: Chromatin was treated with the M.CviPI methyltransferase (Cat # M0227B; New England Bio Labs, Ipswich, MA), which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin. .. Following bisulfite treatment of the M.CviPI-methylated chromatin and subsequent genomic sequencing, we identified endogenous methylation (CpG) and accessibility (GpC) in the same sequencing reaction.

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression
    Article Snippet: Paragraph title: Nucleosome Occupancy and Methylome Sequencing ... This involved harvesting intact nuclei and treating with 200 U GpC methyltransferase M.CviPl (New England Biolabs, Ipswich, MA) for 15 min at 37°C followed by termination of the reaction with an equal volume of 20 mM Tris HCl pH 7.9, 600 mM NaCl, 1% (w/v) SDS, and 10 mM EDTA and overnight digestion with 200 μg/ml Proteinase K (Ambion, Austin, TX).

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: Paragraph title: Nucleosome occupancy and methylome sequencing ... The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C.

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: Next, the bisulfite-treated genomic DNA from a single cell was amplified by a PBAT strategy to obtain sufficient material for sequencing. .. Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM.

    Article Title: Age-associated epigenetic modifications in human DNA increase its immunogenicity
    Article Snippet: 1μg of DNA was methylated in GC Reaction Buffer containing 60 units of GpC Methyltransferase (M.CviPI) and 160 μM S-adenosylmethionine from New England Biolabs (Ipswich, MA) at 37o C for either 4 or 18 hours. .. The GC Methyltransferase methylates all cytosine residues within the double stranded recognition sequence of 5'..GC..3'.

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: A nucleosome occupancy and methylome sequencing (NOMe-seq) assay was performed as described previously ( ) using ∼6 × 105 cells from differentiated 17Abic cultures. .. Intact nuclei were treated with 200 U of the GpC methyltransferase M.CviPI (New England BioLabs) and SAM for 8 min at 37°, followed by an additional 100 U of M.CviPI and SAM for 8 min at 37°.

    Plasmid Preparation:

    Article Title: Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum
    Article Snippet: DNA Isolation and manipulation Plasmid DNA was extracted and purified from E. coli DH5α and ER1821 using an EZ-10 Spin Column Plasmid DNA Miniprep Kit from Bio Basic, Inc. (Markham, ON, Canada). .. Taq DNA polymerase, restriction endonucleases, CpG (M.SssI) and GpC (M.CviPI) methyltransferases, Quick Ligation Kit, and 1 kb DNA ladder were purchased from NEB (Ipswich, MA, USA).

    Software:

    Article Title: A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome
    Article Snippet: Chromatin was treated with the M.CviPI methyltransferase (Cat # M0227B; New England Bio Labs, Ipswich, MA), which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin. .. Each fastq file was aligned to a bisulfite-converted genome (hg19) using BSMAP and processed using the software programs Picard ( http://picard.sourceforge.net ) and SAMtools ( http://samtools.sourceforge.net ).

    Negative Control:

    Article Title: Single-molecule long-read sequencing reveals the chromatin basis of gene expression
    Article Snippet: GpC-specific methyltransferase M.CviPI (NEB) supplemented with 160 µM SAM S-adenosylmethionine was used to methylate spheroplasts at 37°C for 45 min. Genomic DNA was extracted using PCI (phenol:chloroform:isoamyl alcohol, 25:24:1) and purified by a Genomic DNA Clean & Concentrator-10 kit (Zymo Research). .. Native genomic DNA extracted from yeast without M.CviPI treatment was used as the negative control (all cytosines at GpC sites are unmethylated).

    In Vitro:

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: Paragraph title: Preparation and in vitro methylation of single-cell nuclei ... Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM.

    Gel Permeation Chromatography:

    Article Title: A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome
    Article Snippet: .. Chromatin was treated with the M.CviPI methyltransferase (Cat # M0227B; New England Bio Labs, Ipswich, MA), which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin. .. Following bisulfite treatment of the M.CviPI-methylated chromatin and subsequent genomic sequencing, we identified endogenous methylation (CpG) and accessibility (GpC) in the same sequencing reaction.

    Article Title: Single-cell nucleosome mapping reveals the molecular basis of gene expression heterogeneity
    Article Snippet: .. Spheroplasts were washed with 1 M sorbitol three times before treatment with GpC methyltransferase and treated with 30 units of M.CviP (NEB) supplemented with 160 µM S-adenosylmethionine (NEB) at 37 °C for 45 min. ..

    Article Title: Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression
    Article Snippet: .. This involved harvesting intact nuclei and treating with 200 U GpC methyltransferase M.CviPl (New England Biolabs, Ipswich, MA) for 15 min at 37°C followed by termination of the reaction with an equal volume of 20 mM Tris HCl pH 7.9, 600 mM NaCl, 1% (w/v) SDS, and 10 mM EDTA and overnight digestion with 200 μg/ml Proteinase K (Ambion, Austin, TX). .. DNA was subsequently isolated and bisulfite converted using EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA).

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: .. The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C. ..

    Article Title: Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells
    Article Snippet: .. Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM. .. In vitro methylation of single-cell nuclei was performed by incubating the mixture in a thermocycler at 37 °C for 30 min before heat inactivation for 20 min at 65 °C.

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells
    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). .. The suspension was carefully mixed before incubating for 8 min at 37 C after which another 25 ul of enzyme and 0.7 ul of 32 mM SAM were added for an additional 8 min incubation at 37C.

    Article Title: Age-associated epigenetic modifications in human DNA increase its immunogenicity
    Article Snippet: .. 1μg of DNA was methylated in GC Reaction Buffer containing 60 units of GpC Methyltransferase (M.CviPI) and 160 μM S-adenosylmethionine from New England Biolabs (Ipswich, MA) at 37o C for either 4 or 18 hours. .. The GC Methyltransferase methylates all cytosine residues within the double stranded recognition sequence of 5'..GC..3'.

    Article Title: Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum
    Article Snippet: .. Taq DNA polymerase, restriction endonucleases, CpG (M.SssI) and GpC (M.CviPI) methyltransferases, Quick Ligation Kit, and 1 kb DNA ladder were purchased from NEB (Ipswich, MA, USA). .. Pfu DNA polymerase and RNase A were purchased from Bio Basic, Inc. (Markham, ON, Canada).

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: .. Intact nuclei were treated with 200 U of the GpC methyltransferase M.CviPI (New England BioLabs) and SAM for 8 min at 37°, followed by an additional 100 U of M.CviPI and SAM for 8 min at 37°. .. Following proteinase K treatment, genomic DNA was extracted and bisulfite converted using an Epitect bisulfite kit (Qiagen) according to the manufacturer's protocol.

    Article Title: Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
    Article Snippet: .. Taking advantage of the fact that mammalian cells lack GpC methylation, intact nuclei are incubated with the GpC methyltransferase M.CviPI, which methylates GpC dinucleotides not associated with nucleosomes or tightly bound transcription factors while leaving nucleosomal or transcription factor-occupied sites unmethylated. .. Following bisulfite conversion, patterns of GpC and CpG methylation are assessed, allowing the determination of both nucleosome occupancy and endogenous methylation status for individual DNA strands.

    Article Title: Single-molecule long-read sequencing reveals the chromatin basis of gene expression
    Article Snippet: .. GpC-specific methyltransferase M.CviPI (NEB) supplemented with 160 µM SAM S-adenosylmethionine was used to methylate spheroplasts at 37°C for 45 min. Genomic DNA was extracted using PCI (phenol:chloroform:isoamyl alcohol, 25:24:1) and purified by a Genomic DNA Clean & Concentrator-10 kit (Zymo Research). .. We denote the above mentioned genomic DNA that undergoes in vivo spheroplast methylation as the target sample of MeSMLR-seq.

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

    DNA Methylation Assay:

    Article Title: Age-associated epigenetic modifications in human DNA increase its immunogenicity
    Article Snippet: Global methylation of DNA from aged and young subjects was determined using the Methylamp™ Global DNA Methylation Quantification Kit from Epigentek (Brooklyn, NY), as per the manufacturer's protocol. .. 1μg of DNA was methylated in GC Reaction Buffer containing 60 units of GpC Methyltransferase (M.CviPI) and 160 μM S-adenosylmethionine from New England Biolabs (Ipswich, MA) at 37o C for either 4 or 18 hours.

    Concentration Assay:

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells
    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). .. The suspension was carefully mixed before incubating for 8 min at 37 C after which another 25 ul of enzyme and 0.7 ul of 32 mM SAM were added for an additional 8 min incubation at 37C.

    Cell Counting:

    Article Title: Single-cell nucleosome mapping reveals the molecular basis of gene expression heterogeneity
    Article Snippet: Spheroplasts were washed with 1 M sorbitol three times before treatment with GpC methyltransferase and treated with 30 units of M.CviP (NEB) supplemented with 160 µM S-adenosylmethionine (NEB) at 37 °C for 45 min. .. Spheroplasts were then diluted based on original cell count to 0.6 cells per microliter.

    Lysis:

    Article Title: DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells
    Article Snippet: Briefly, cell pellet was first lysed in 1× lysis buffer (part of the NOMe-Seq Kit, Active motif #54000) with Protease inhibitor and PMSF (part of the NOMe-Seq Kit, Active motif #54000) on ice for 1 h with intermittent vortex. .. The nuclei was released, washed with DPBS, and spiked in with 0.1% unmodified lambda DNA (ThermoFisher #SD0011), and then subjected to the 15 U M.CviPI GpC Methyltransferase (NEB #M0227L) treatment for 1 h at 37 °C, supplemented with 160 nM fresh SAM (NEB #B9003S), then followed by a boost with an additional 15 U M.CviPI and 160 nM fresh SAM for another 2 h at 37 °C.

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells
    Article Snippet: Nuclei were collected by centrifuging the cell suspension for 5 min at 800x g at 4 C and washed twice with cold lysis buffer without detergent. .. 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).

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    New England Biolabs gpc methyltransferase m cvipl
    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 <t>GpC</t> 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.
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    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

    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