Seqcap epi enrichment system

Browser extensible data (BED) files for SeqCap were created for all genes of interest using the table browser in the University of California, Santa Cruz (UCSC) Genome Browser Gateway[36 ]. For promoter regions, we chose 2000 bp upstream of the gene. Genome locations were converted from hg19 to hg38 using the UCSC Genome LiftOver tool[37 ].
A total of 90 μL of DNA (normalized to 50ng/uL, 260/280 > 1.8) from each participant was sent to the Functional Genomics Laboratory at the University of California, Berkeley for DNA methylation analysis by the Roche Nimblegen, SeqCap Epi Enrichment System, following company protocol[14 ]. Raw sequencing data was then processed at the UC Davis Bioinformatics Core. Briefly, pair-end 151 bp raw sequencing reads were subjected to adapter removal by scythe-0.993. Bases that have qualities lower than 30 were trimmed using sickle-1.33, and trimmed reads that are less than 30 bp long were not considered for downstream analysis. The reads that have passed the above quality control were aligned to the human genome (version GRCh38), using BSseeker2[38 (link)]. Bowtie2 [39 (link)] was used as the underlying aligner. Methylation status was identified using a built-in function in BSseeker2. The methylation results for the regions that include the selected genes, as well as 20 K bp up- and down-stream were aggregated using bedtools-2.25.0[40 (link)].

Bone marrow samples were subjected to Ficoll density gradient separation (Sigma-Aldrich, St. Louis, MO, USA) to enrich for mononuclear cells, followed by DNA purification using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). MethylC-capture sequencing was performed as previously described [14] (link). Briefly, bisulfate-converted DNA fragments were amplified, a sequencing library was constructed using the SeqCap Epi enrichment system (Roche NimbleGen, Madison, WI, USA), and the DNA was sequenced using a HiSeq2500 system (Illumina, San Diego, CA, USA).