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Bisulfite sequencing for poly(A)+ RNA

ABSTRACT: Bisulfite sequencing for poly(A)+ RNA

PROVIDER: PRJNA625666 | ENA |

REPOSITORIES: ENA

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Project description:We applied the solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of 1 million CpGs in more than 21,408 CGIs and 15,946 transcriptional regulatory regions. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. In this study, we generated genome-wide, single-base resolution DNA methylation maps in three of the most commonly used breast cancer cell lines.Differentially methylated regions (DMRs) were identified in the 5?-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X chromosome among the three cell lines. The single CpG resolution methylation maps of many known tumor suppressor genes were also established in the three cell lines. Here we present a novel approach that combines solution-phase hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in targeted CGI and promoter regions. We designed 51,466 single strand DNA oligonucleotides (160-mer) which target 23,441 CGIs and the transcription start sites of 19,369 known genes in the human genome. The synthetic long DNA oligonucleotides were converted into biotinylated RNA probes for solution-phase hybridization capture of target DNA. The captured genomic DNA was treated with sodium bisulfite, amplified by PCR and sequenced using Illumina GA IIx sequencer.

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