ChIP-slot blot

The control, USP7-KO and FLAG-USP7-expressing USP7-KO HeLa cells were synchronized to S phase and labeled with BrdU (Sigma) for 20 min. The cells were cross-linked with 1% formaldehyde for 15 min before neutralization with 0.125 M glycine. The cells were lysed by sonication in ChIP Lysis buffer (25 mM Tris-HCl, pH 8.0, 0.1% SDS, 1 mM EDTA, 1× protease inhibitor cocktail), and soluble cell extracts were recovered after centrifugation at 12,000 rpm and 4 °C for 20 min. The supernatant was incubated with DNMT1 antibody overnight at 4 °C on a rotator. Chromatin-antibody complexes were isolated with 20 μL of Protein G beads blocked by sperm DNA and bovine serum albumin. After extensive washing, protein/DNA complexes were eluted from the beads in Elution buffer (50 mM Tris-HCl, pH 8.0, 1% SDS, 10 mM EDTA) at 65 °C overnight. Immunoprecipitated DNA was purified by phenol/chloroform extraction, and analyzed by slot blot using BrdU antibody.