title: RRBS of homo sapiens: PBMC
design: Bisulfite-converted MspI-restricted genomic fragments were molecularly indexed and sequenced using an in-house developed RRBS method [PMID 37494662]. Genomic DNA isolated from PBMCs was fragmented using MspI restriction enzyme. The fragmented DNA were ligated with two adaptors, adapter-A and adapter-B, using Blunt/TA Ligase Master Mix (NEB). This method utilized two different adaptors, each with a CpG overhang in one end. Adaptor-A contained a molecular barcode and the binding site for Illumina R1 sequencing primer, while Adaptor-B carried the binding site for Illumina R2 sequencing primer. All cytosines on the Illumina primer binding sites of the adaptors were protected from bisulfite conversion. After ligation, the fragments underwent a fill-in-step to remove unligated adaptor strand. During the fill-in-step process, dCTP was replaced with methyl-dCTP. Bisulfite conversion was then performed using the EpiTect Fast DNA Bisulfite Kit (Qiagen). The converted fragments were amplified by two rounds of PCR to generate sequencing-ready libraries. The first round of PCR was performed using primers P1 and P2 for 10 cycles under following conditions: 95 C for 30 seconds, 60 C for 30 seconds, and 72 C for 30 seconds. The second round of PCR, using index primer idxP1 and idxP2, was conducted for an additional 10 cycles under following conditions: 95 C for 30 seconds, 60 C for 30 seconds, and 72 C for 30 seconds. Finally, massive parallel sequencing was performed on Illumina Hiseq X platform with 309 bp paired-end reading