GSM7893299: R1-CBX7-rep2; Mus musculus; ChIP-Seq
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 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 hour before incubating with antibodies overnight at 4°C. Antibody–chromatin complexes were washed with RIPA150 (50 mM Tris-HCl pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate and protease inhibitors) once, RIPA500 (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate and protease inhibitors) twice, RIPA-LiCl (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 0.5 M LiCl and protease inhibitors) twice and 1×TE twice. The precipitations were eluted by 200μl NLB (50 mM Tris-HCl pH 8.0, 10 mM EDTA and 1% SDS) at 65°C. Reverse crosslink was performed at 65°C overnight. The DNA libraries were purified with 1 volume AMpure beads and sequenced on Nova-seq6000 with paired-end 150 bp (Illumina).