GSM4143976
GSM4143976: HUtSMC-crCtrl-INPUT-Rep-3; Homo sapiens; ChIP-Seq
16% paraformaldehyde was added to full media-containing HUtSM cell plates to a final concentration of 1% paraformaldehyde and incubated at room temperature for 10min and then quenched with 0.125M glycine for 10min at room temperature with rotation. Cross-linked cells were then incubated in ChIP lysis buffer 1 (50mM HEPES-KOH [pH 7.6], 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100, 1X Protease inhibitor cocktail [Roche, cat # 4693132001]) followed by end-over-end rotation at 4°C for 15min and then low speed centrifugation at 4°C. Samples were resuspended in ChIP lysis buffer 2 (10mM Tris-HCl [pH 8.0], 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 1X Protease inhibitor cocktail [Roche, cat # 4693132001]) followed again by end-over-end rotation at 4°C for 15min and sample recovery by low speed centrifugation at 4°C. Samples were then resuspended in ChIP lysis buffer 3 (10mM Tris-HCl [pH 7.5], 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% sarkosyl) and sonicated in an ice water bath (Misonix, setting 6 [~6 W power output], 12 cycles of 15sec on and 45sec off). Triton X-100 was added to a final concentration of 1% and samples were centrifuged at 20,000 x g at 4°C for 20min, with recovery of chromatin-containing supernatant. Solubilized chromatin was incubated overnight with antibody at 4°C with end-over-end rotation. KAPA Hyper Prep kit (Kapa Biosystems, cat # KK8502) was used for end-repair, A-tailing, and adapter ligation with TruSeq index adapters, all according to manufacturer's instructions. A 0.6X-0.8X AMPure XP bead (Beckman Coulter, cat # A63881) size selection was carried out after adapter ligation to enrich for sub-nucleosomal DNA fragments followed by 12 cycles of PCR amplification.
GEO
GSM4143976
GEO Accession
GSM4143976