GSM1971016: RRBS_Control_HCT116; Homo sapiens; Bisulfite-Seq
Total DNA of HCT116 cell line with or without H2O2 treatment was extracted by proteinase K/phenol. For each library, 500 ng of genomic DNA was digested overnight with MspI (20 units). After purification, the digested products were blunt-ended. Then dA was added, followed by methylated-adapter (Illumina, CA, USA) ligation. To obtain DNA fractions in 40-120 bp and 120-220 bp range of MspI-digested products, two ranges (160-240 bp and 240-340 bp) of adapter-ligated fractions were excised from 2% agarose gel. The size-selected DNA was bisulfite-treated for two rounds using EpiTect Bisulfite Kit (Qiagen, Venlo, Netherlands) following the manufacturer’s standard protocol. Finally, libraries were generated by amplification with HiFi HotStart Uracil+ ReadyMix (KAPA, MA，USA) and Illumina Multiplexing PCR Primers. Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) and RT-PCR were used to guarantee the quality and quantity of RRBS library. After RRBS library construction, Illumina HiSeq 2000 was used to sequence those libraries according to the manufacturer’s protocol.