GSM7916631: ChIP-seq_RAW264.7_macroH2AS139A_HA_rep1; Mus musculus; ChIP-Seq
The cells were crosslinked in adherent conditions with 1% formaldehyde for 15 min at room temperature and quenched with glycine to a final concentration of 125 nM. Fixed cells were lysed in nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitors) to extract nuclei and sheared by sonication to 200-500-bp-sized fragments. Sheared chromatin was incubated with rabbit anti-HA antibodies (Abcam, ab9110, 3 μg) and protein A/G-Dynabeads (Life Technology, 10 μl) for 12 hours at 4 ℃. The beads were washed with RIPA150 (50 mMTris-HCl, pH 8.0,150 mM NaCl, 1 mM EDTA, pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), RIPA500 (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl,1 mM EDTA, pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), RIPA-LiCl (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 0.5 M LiCl) and TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), respectively. The beads were resuspended with NLB buffer (10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.5% SDS). After de-crosslinked at 65 ℃ for 6 hours, DNA fragments were digested with RNase A and proteinase K. Purified DNA fragments were generated libraries followed by high throughput sequencing using Illumina Novaseq6000. Purified DNA fragments were generated libraries followed by high throughput sequencing using Illumina Novaseq6000.