title: GSM5554963: PERK_KO_M2_2; Mus musculus; ChIP-Seq
library_construction_protocol: Five millions of cells were fixed with 1% formaldehyde in media at 1x10^6 cells/mL for 10 min at room temperature with constant agitation. Fixation was stopped and quenched with 125 mM glycine for 5 min on ice. After one wash with PBS, cell pellets were snap-frozen with liquid nitrogen and store at -80°C until nuclei preparation. Nuclei were isolated using Lysis buffer (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100) and were washed once with Wash buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). The pellets were resuspended with Shearing buffer (10mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS) and the chromatin were sheared using BioruptorPico (Diagenode) for 14 cycles (30s on; 30s off) at 4°C. 2.5 µg chromatin was diluted to a final of 300 µL RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate) and incubated with 2.5 µg rabbit monoclonal anti-H3K27me3 antibody (CST, clone C36B11, lot 19) overnight at 4°C with constant rotation. For immunoprecipitation, the samples were incubated with 10µL pre-cleared Protein A Magnetic Beads (ThermoFisher) for 2h at 4°C. The beads were washed twice with RIPA buffer, once with High salt buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS), once with LiCl buffer (50 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP40, 1% sodium deoxycholate), and finally once with TE buffer (10 mM Tris-HCl pH 8.0, 1mM EDTA). All washes were incubated for 5 mins at 4°C with constant rotation. After last wash, beads were resuspended with 200L elution buffer (100 mM NaHCO3, 1% SDS) with 0.5 mg/mL RNaseA (Qiagen), and incubated at 65°C on a thermoshaker for 20 min at 1200 rpm. For decrosslinking, proteinase K (0.5 mg/mL; Ambion) and NaCl (200 mM) were added into the eluted samples and incubated in thermocycler at 65°C overnight. The samples were purified by using Zymo PCR purification kit (Zymo). Sequencing libraries were prepared using NEB Ultra II kits according to the standard protocol (NEB).