Reduced representation bisulfite sequencing data of mouse genome
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE76238 | GEO | 2016/02/26
SECONDARY ACCESSION(S): PRJNA306687
REPOSITORIES: GEO
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Project description:Reduced representation bisulfite sequencing (RRBS) has been proven a powerful method in DNA methylome profiling. Since the initial development of this method, the RRBS protocol has been modified in order to optimize it for genomic coverage, starting material, and library-construction throughput, which has resulted in new methods such as enhanced RRBS (ERRBS), double-enzyme RRBS (dRRBS), gel-free and multiplexed RRBS (mRRBS), and single-cell RRBS (scRRBS). However, each of these methods has failed to address PCR-derived duplication artifacts, which can bias the results of DNA methylation analyses. To overcome the aforementioned complication, we developed quantitative RRBS (Q-RRBS), a method in which unique molecular identifiers (UMIs) are used to eliminate PCR-induced duplication. By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.
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Project description:Differential hyper- and hypo-methylation regions in G0 versus G4/G5 CMP The goal of this study is to evaluate changes in CpG methylation profilings of telomere dysfunctional common myeloid progenitor cells (CMP) as compared to their wild type controls Genomic DNA was extracted from sorted CMP populations isolated from 3 pools of G0 or 2 pools of G5 mice using UltraPure Phenol:Chloroform:Isoamyl Alcohol according to manufacturer’s instructions (Life Technologies). 14,000 to 30,000 cells were available for each sample, resulting in a minimum of 45ng of DNA. Genome-wide DNA methylation profiling was performed by RRBS. Library preparation and sequencing were performed at the UT MD Anderson Cancer Center’s DNA Methylation Analysis Core and Sequencing and Microarray Facility, according to published protocols. RRBS sequencing data were aligned and methylation was called using Bismark v0.7.119. In brief, bisulphite-treated DNA was aligned to UCSC Genome Browser mm10 reference genome using Bowtie. In total 29-38 million reads were generated per sample with alignment rates around 63%. Next, MethylKit10 implemented with Fisher’s exact test was used to compare the cytosine methylation profiles of G0 and G5 CMP. Gene promoter regions were calculated based on RefSeq gene annotations with regions starting 1 kb upstream of the annotated transcription start site (TSS) and extending 500 base pairs downstream of TSS. Exons, introns, and CpG islands coordinates were collected from the UCSC Genome Browser mm10 version.
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