GSM9689860: MDA-MB-231, CT7311-treated, RPA2-ChIP (biological replicate 1); Homo sapiens; ChIP-Seq
Cells treated with either DMSO or CT7311 were crosslinked with 1% formaldehyde for 5 min at room temperature (RT), followed by quenching with 0.125 M glycine for 5 min at RT. Nuclei were extracted using a Lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100, and protease inhibitors) and then sonicated to an average DNA length 200 bp in Shearing buffer (10 mM Tris-HCl [pH 8.1], 0.1% SDS, 1 mM EDTA, and protease inhibitors) using Covaris E220 sonicator. The sonication settings were as follows: 5% duty cycle, 140 Watts peak power, 200 cycle per burst, for 10 min. Following sonication, the chromatin was supplemented with 1% Triton X-100 and 150 mM NaCl, then centrifuged. For each ChIP reaction, chromatin from ~ 10 million cells was incubated with an RPA32 antibody (FORTIS life science) at 4°C overnight. The following day, protein A/G agarose beads were added and incubated for 2 hours with rotation. The beads were subsequently washed with 4 times with the following buffers: Low salt wash buffer (20 mM HEPES-KOH [pH 7.5], 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, protease inhibitors), High salt wash buffer (20 mM HEPES-KOH [pH 7.5], 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, protease inhibitors),  Low LiCl wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 1% IGEPAL CA-630, 1% Sodium deoxycholate, protease inhibitors), TE buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA). ChIP and input samples were treated with RNase A and Proteinase K, and reverse crosslinking was performed by incubating at 65 °C overnight. DNA was purified with phenol-chloroform extraction and isopropanol precipitation. ChIP-seq libraries were prepared by Azenta (Genewiz, NJ, USA).