GSM3667627: PT967-LEIO-RNAPII; Homo sapiens; ChIP-Seq
Myometrium and leiomyoma tissue samples were cryopulverized into a fine powder and immediately formaldehyde cross-linked with 1% formaldehyde for 10min at room temperature with rotation and then quenched with 0.15M glycine for 10min at room temperature with rotation. Cross-linked tissue samples were dounce homogenized 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 MNase digestion buffer (20mM Tris-HCl [pH 8.0], 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS, 125U MNase [Worthington, cat # LS004798]) and incubated at 37°C until 75% mononucleosomal chromatin profile is observable by gel electrophoresis of purified MNase digested DNA. Digestion was quenched with MNase quenching buffer (10mM EDTA, 20mM EGTA) after which samples were briefly sonicated (Misonix, setting 6 [~6 W power output], 3 cycles of 15sec on and 45sec off). Collagen and cell debris was removed by centrifugation at 20,000 x g at 4°C for 20min. 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 1.1X-1.8X AMPure XP bead (Beckman Coulter, cat # A63881) size selection was carried out after adapter ligation to enrich for sub-nucleosomal DNA fragments followed by 10-12 cycles of PCR amplification. For H3K27Ac ChIP, a 0.6X-1.0X AMPure XP bead size selection was performed to enrich for mono-nucleosomal DNA fragments.