GSM4502223: KRp2-112; Homo sapiens; Bisulfite-Seq
The plasma was transferred to 2-mL microcentrifuge tube and centrifuged at 16,000g for 10 minutes at 4°C to remove any cellular debris. Circulating cell-free DNA (10–50 ng) was extracted from 1-2 ml plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen) and quantified using the Quant-iT high-sensitivity Picogreen double-stranded DNA Assay Kit (Invitrogen by Thermo Fisher Scientific) according to manufacturer instructions. For targeted methylation sequencing, 10 ng plasma cell-free DNA was first bisulfite treated using the ZYMO Gold Kit per the manufacturer's protocol. We adapted Swift Bioscience Methyl-Seq library preparation kit to generate individual libraries incorporating 13 PCR cycles and overnight ligation. Custom targeted CpG methylation probes were designed using Roche Nimblegen target capture kit, Custom SeqCap Epi Choice 30 MB. Libraries were quantified using Quant-iT high-sensitivity Picogreen double-stranded DNA Assay Kit before equimolarly pooling 10 individual libraries per capture consisting of 2 ug total DNA. Hybridization and capture were performed using VK SeqCap Epi Reagent Kit Plus and SeqCap EZ hybridization/wash kit from Roche Nimblegen following manufacturer recommendations. For blocking, we used universal blocker from IDT technologies. We sequenced the pooled libraries on Illumina NovaSeq S4 using paired-end 100-base-pair reads incorporating 150 individual libraries per lane.