DPC-seq of HeLa cells: WT NMS873 0h 3rd
HeLa cells were lysed in SDS buffer (2% SDS; 20 mM Tris, pH 7.5) followed by sonication using Covaris M220 system (5% duty factor, 200 cycles per burst, 16 min at 25C in microTUBE). The sonicated samples were incubated with KCl buffer (200 mM KCl; 20 mM Tris, pH 7.5) on ice for 5 min. After centrifugation at 20,000 g for 5 min at 4C, the pellets were washed five times by addition of KCl buffer followed by incubation at 55C for 5 min. After centrifugation, the pellets were dissolved in KCl buffer with 0.04 mg/ml Proteinase K and incubated at 55C for 3 h. After centrifugation at 20,000 g for 5 min at 4C, the supernatant was purified using MinElute PCR Purification Kit (QIAGEN). Purified DNA was subjected to preparation of next-generation sequencing libraries using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). The library quality and quantity were verified by Multina (SHIMADZU) or 4150 TapeStation (Agilent).