GSM7850113: MCF7_H3K27ac; Homo sapiens; ChIP-Seq
Post-confluent cells were fixed with 1% formaldehyde for 10 min, followed by quenching with 125mM glycine and washed with cold TBS (20mM Tris-HCl, 150mM NaCl, pH 7.4) twice. Cells were lysed in cell lysis buffer (10mM Tris-HCl, 10mM NaCl, 0.5% NP-40, pH7.5) and then incubated on ice for 10 mins. Lysates spinned down and resuspended in 500 μl MNase digestion buffer (20mM Tris-HCl, 15mM NaCl, 60mM KCl, 1mM CaCl2, pH 7.5) with MNase (New England, M0247S, 0.25U/1000 cells). After 20 minutes of incubation at 37°C with continuous mixing, digestion was stopped by addition of 500 μl of 2X STOP/ChIP buffer (100mM Tris-HCl, 20mM EDTA, 200mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate, pH8.1). Samples were sonicated for 7 cycles (30 secs on/30 secs off) with 40% amplitude (Branson Digital Sonifier) and then centrifuged at 12,000rpm for 15min at 4°C to remove insoluble debris. Apart from input samples (1% volume for each ChIP reaction) kept for reverse-crosslinking, lysates were incubated with antibodies (antibody information: see table attached) at 4°C overnight. Antibody-bound complexes were then captured by incubation with Protein A sepharose CL-4B beads (Amersham) for rabbit antibodies or Protein G sepharose 4B beads (Invitrogen) for mouse antibodies (both 20μl per 1mL lysate) for 4 hr at 4 °C. The beads were washed sequentially with STOP/ChIP buffer (50mM Tris-HCl, 10mM EDTA, 100mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, pH8.1), high salt buffer (50mM Tris-HCl, 10mM EDTA, 500mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, pH8.1), LiCl2 buffer (10mM Tris-HCl, 0.25M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, pH8.0), and TE buffer (50mM Tris-HCl, 10mM EDTA, pH 8.0) (twice). Bound DNA was eluted and reverse-crosslinked at 65°C overnight in ChIP elution buffer (10mM Tris-HCl, 150mM NaCl, 10mM EDTA, 5mM DTT, 1% SDS, pH 8.0). After treatment with RNase A (0.2mg/ml, 37°C, 1h) and proteinase K (2mg/ml, 37°C, 2h), DNA used for sequencing was purified using a MiniElute PCR purification kit (Qiagen), and DNA used for qPCR was purified using a QIAquick PCR purification kit (Qiagen). DNA quality was assessed using an Agilent Bioanalyzer, and quantified using a Qubit Fluorometer (Invitrogen). ChIP-seq libraries were generated using DNA SMART ChIP-seq kits (Takara Bio) according to the manufacturer's instructions. After size selection (for 250-600bp) by AMPure XP beads (Beckman Coulter), the library was quality was evaluated using the Bioanalyzer and sequenced on Illumina NovaSeq SP (100bp two ends) and Illumina NextSeq High (75 bp single end).