Journal: bioRxiv

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

doi: 10.1101/061739

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

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.

Techniques: Gel Permeation Chromatography, DNA Methylation Assay

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: 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).

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