title: GSM8768492: KO-52, Tm, INPUT; Homo sapiens; ChIP-Seq
library_construction_protocol: HPC-7 cells were cross-linked with 1% formaldehyde and lysed (1% Triton, 0.1% sodium deoxycholate, 0.1% SDS, 0.2M NaCl, 10mM Tris pH 7.5, 10mM EDTA and 1X protease inhibitor cocktail (Roche 11836170001)). Sonication was performed in the same buffer with 0.5% SDS in a Bioruptor (Diagenode) for 60 cycles (30s ON 30s OFF), vortexing every 20 minutes. Sodium butyrate 5mM was added to the buffers in the cells for the H3K27ac ChIP to inhibit deacetylases. For H3K4me1 ChIP-seq, DNA was digested in 50mM Tris pH 7.5, 4mM MgCl2 and 2mM CaCl2 with 10U micrococcal nuclease (Thermo Scientific EN0181) for 15 min at 22ºC. For ATF4 ChIP, KO-52 cells were pre-treated with 2µg/mL tunicamycin for 4h and additionally crosslinked with 1mM DSG (A35392, ThermoScientific) for 45 minutes. Lysates were sonicated for 30 min in lysis buffer (0.1% SDS). Lysates were incubated with 4µg of H3K27ac antibody (Abcam ab4729), 8µg of anti-ERa (sc-8002X), 3µg anti-H3K4me1 (Diagenode, C15410194), 22µL anti-CEBPA (8178, Cell Signaling) or 10 µL  anti-ATF4 (11815, Cell Signaling) overnight at 4ºC. Antibody-bound chromatin was pulled down with protein G Dynabeads (Invitrogen 10003D). Samples were washed, RNase-treated and reverse cross-linked by incubation at 65°C in 1% SDS, 0.1M NaHCO3 and proteinase K. DNA was purified using ChIP DNA Clean & Concentrator Kit (Zymo D5205). Libraries were prepared using the NEBNext Ultra DNA Library Prep kit (E7645, New England Biolabs).