GSM4648966: DMSO_AR-IP_Rep1; Homo sapiens; ChIP-Seq
10 million cells were fixed using 1% formaldehyde (Thermo Fisher Scientific, BP531-25) for 10 minutes at room temperature followed by quenching with 125 mM glycine (Sigma-Aldrich, #50046). Cells were rinsed twice with PBS and resuspended in 1 mL lysis buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche, #11836170001). Chromatin was sheared to 200-500 base pairs using a Covaris E220 sonicator and cleared by centrifugation for 15 minutes at 19,000 x g. Antibodies (AR, 9 μg, Abcam, ab74272; H3K27ac, 1 μg, Diagenode, C15410196) were incubated with 40 μL of protein A/G Dynabeads (Thermo Fisher Scientific, #10002D, #10003D) for at least 6 hours at 4°C before overnight incubation at 4°C with sonicated chromatin. Chromatin-bead complexes were washed 5 times with 1 mL LiCl wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate) and rinsed twice with 1 mL TE buffer (10 mM Tris pH 7.5, 0.1 mM EDTA). Immunoprecipitated chromatin was resuspended in 100 μL elution buffer (100 mM NaHCO3, 1% SDS) and treated with RNase A (Thermo Fisher Scientific, #12091021) for 30 minutes at 37°C. Crosslinks were reversed in the presence of proteinase K (Thermo Fisher Scientific, #25530049) for 16 hours at 65°C, and the eluted DNA was purified using a MinElute PCR Purification Kit (QIAGEN, #28006). ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Preparation Kit (NEB, #E7645).
GEO Accession
GSM4648966