H2B-DPC-seq of HeLa cells: CSB-KO 4h 1st
HeLa cells were incubated in EBC buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 0.5% NP-40; 5 mM EDTA; 0.5 mM EGTA) supplemented with protease inhibitor cocktail (Roche) for 20 min on ice. After centrifugation at 10,000 g for 5 min at 4C, the pellets were dissolved in incubation buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; 5 mM EDTA; 0.5 mM EGTA; 0.1% BSA) supplemented with protease inhibitor cocktail (Roche), and sonicated using Covaris M220 system (5% duty factor, 200 cycles per burst, 16 min at 7C in microTUBE). After centrifugation at 20,000 g for 10 min at 4C, the supernatant was incubated with anti-H2B (ab1790, abcam) antibodies and Protein A and G magnetic beads (Bio-Rad) for 12-16 h on a rotator at 4C. After extensive washing, the beads were incubated with SDS buffer (2% SDS; 20 mM Tris, pH 7.5) for 15 min at 30C. Then, the eluted supernatant was 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 MGIEasy Universal DNA Library Prep Set (MGI). The library quality and quantity were verified by Multina (SHIMADZU) or 4150 TapeStation (Agilent).