title: GSM5333141: MCF7-P E2 ER rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: Breast cancer cells were grown to 85% confluence in 15-cm plates and cross-linked with 1% formaldehyde (10 min) followed by quenching (125 mM glycine). Cells were washed in cold PBS and harvested by cell scraper in cold PBS with protease inhibitors (Roche). Cells were centrifuged at 3,220g for 5 min, snap-frozen, and stored at -80°C at 1×10^7 cells per pellet. Pellets were resuspended in cytosolic and then nuclear lysis buffer, and chromatin was sheared at 4°C using a water-bath sonicator (Bioruptor, Diagenode) for 20 min at high output (30 s on, 30 s off). Sonicated lysates were cleared by centrifuging at 20,000g for 10 min and diluted 4 times before pre-incubation with protein-A/G beads (Santa Cruz) for 30 min at 4°C. ChIP was performed by overnight incubation at 4°C with antibodies against human ER (Santa Cruz Biotechnology, sc-543X) and Drosophila melanogaster histone variant H2Av (Active Motif, # 61752) to pull down spiked-in chromatin for normalization control, followed by an additional 1 h incubation with protein-A/G beads. Beads were washed consecutively with low and high salt wash buffer, once with LiCl wash buffer, and once with TE buffer. DNA was eluted in elution buffer (50 mM NaHCO3 and 1% SDS) and then supplemented with 300 mM NaCl. Cross-links were reversed overnight at 67°C. RNA was digested at 37°C with 0.1 mg/ml RNase A for 30 min. DNA was purified with a PCR purification kit (Qiagen). Indexed libraries were prepared from ChIP DNA using the KAPA Hyper Library Preparation Kit (Kapa Biosystems). Libraries were amplified by 12 cycles of PCR, and then assessed for size distribution using the 4200 TapeStation High Sensitivity D1000 ScreenTape (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher). The indexed libraries were multiplexed, 10 libraries per pool. The pool was quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems) and then sequenced on the Illumina HiSeq 2000.
attributes.tag: GEO Accession
attributes.value: GSM5333141