Project description:We report the analysis of DNA methylation in mouse chromaffin cell lines using reduced representation bisulfite sequencing (RRBS). We compared DNA methylation profiles of cell lines with or without a knock-out of Sdhb gene, showing that Sdhb disruption results in a hypermethylator phenotype. Reduced representation bisulfite sequencing of 4 mouse chromaffin cell samples (2 Sdhb wild-type and 2 Sdhb knock-out).
Project description:To assess variation and inheritance of genome-wide patterns of DNA methylation simultaneously in humans, we applied reduced representation bisulfite sequencing (RRBS) to somatic DNA from six members of a three-generation family. Reduced representation bisulfite sequencing was applied to genomic DNA from leukocytes of 6 family members and two unrelated individuals.
Project description:DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging due to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called Laser Capture Microdissection-Reduced Representation Bisulfite Sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of CpG Islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared to adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development. Laser capture microdissection-reduced representation bisulfite sequencing and reduced representation bisulfite sequencing on human blood leukocyte, human endometrial tumor, mouse liver tissue, and mouse normal and neoplastic adrenal tissue
Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells. 12 samples, reduced representation bisulphite treatment: 4 replicates each for bisulphite (BS), oxidative BS (oxBS) and reduced BS (redBS) for the detection of 5mC, 5hmC and 5fC. Mouse (strain B6C) embryonic stem cells.
Project description:DNA methylation plays critical roles in gene regulation and cellular specification without altering DNA sequences. The wide application of reduced representation bisulfite sequencing (RRBS) and whole genome bisulfite sequencing (bis-seq) opens the door to study DNA methylation at single CpG site resolution. One challenging question is how best to test for significant methylation differences between groups of biological samples in order to minimize false positive findings. Current methods to analyze genome-wide bisulfite sequencing data use a smoothing approach or a simple statistical test based on the binomial distribution. Comparative DNA methylation profiling in AML blasts and normal CD34(+) control cells
Project description:We report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput Reduced Representation Bisulfite Sequencing (RRBS) and single-molecule-based sequencing, we generated DNA methylation maps covering the vast majority of CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for murine embryonic stem (ES) cells, ES-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of ES-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumors. More generally, the results establish RRBS as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine. Keywords: High-throughput Reduced Representation Bisulfite Sequencing (RRBS), Illumina, cell type comparison Reduced representation bisulfite sequencing (MspI,~40-220bp size fraction) of 18 murine cell types. Raw sequence data files for this study are available for download from the SRA FTP site at ftp://ftp.ncbi.nlm.nih.gov/sra/Studies/SRP000/SRP000179