GSM7316335: ChIP-seq_CBX7_T8_rep1; Mus musculus; ChIP-Seq
For RING1B, RYBP, HA and FLAG ChIPs, mESCs were fixed with 2.5 mM ethylene glycol- bis(succinimidylsuccinate) (EGS) for 45 min, then washed twice with PBS and fixed again with 1% formaldehyde for 10 min. For the H3K27me3 and RING1B- ChIP experiments , mESCs were only fixed with 1% formaldehyde for 10 min. Fixed mESCs were resuspended in nuclei lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS and protease inhibitors) and sheared to 300- to 500-bp-sized fragments. 100 μl of sheared chromatin was diluted using 900 μl ChIP dilution buffer (50 mM Tris-HCl (pH 8.0), 0.167 M NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate and protease inhibitors). Diluted chromatin was pre-cleared with Protein A/G beads 1 h before incubating with antibodies overnight at 4 °C. Antibody–chromatin complexes were washed with RIPA150 (50 mM Tris-HCl (pH 8), 0.5 M NaCl, 1 mM EDTA (pH 8), 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate and protease inhibitors) once, RIPA500 twice, RIPA-LiCl (50 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8), 1% NP-40, 0.7% sodium deoxycholate, 0.5 M LiCl and protease inhibitors) twice and 1× TE twice.