GSM5961923: H3Q5ser-HeLa-1_S2_L001; Homo sapiens; ChIP-Seq
Adherent HeLa S3 cells were crosslinked with 1% formaldehyde for 10 minutes, and the reaction was stopped with 0.125 M glycine for 10 minutes. Chromatin was sheared by sonication (Covaris S220) to 200-800 bp in ChIP lysis buffer (50 mM Hepes, pH 7.4, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 0.5 mM PMSF). 5-10 ug of sonicated chromatin were incubated with H3K9me3 antibody (Active Motif, #39161) and H3Q5ser antibody (Millipore Sigma, #ABE1791) at 4°C for 4 h, then incubated with washed Protein A/G Dynabeads (Invitrogen, #10001D) at 4°C for 2 h. The beads were washed once with ChIP Lysis Buffer, High Salt Buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate), LiCl Buffer (20 mM Tris, pH 7.4, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and TE Buffer (10 mM Tris PH8.0, 1 mM EDTA) in sequence. The beads were resuspended with Elution Buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1% SDS) and incubated at 65°C for overnight. The crosslink-reversed ChIP product was then treated using RNase A and Proteinase K. The final sample was extracted using QIAquick PCR Purification Kit (Qiagen). ChIP-seq library is prepared using a SWIFT Accel-NGS® 2S DNA Library Kit