title: GSM9405925: V5(MORC3) ChIP-Seq in MORC3.AID.V5 SPI1.dTAG cells treated with dTAGV1, Replicate 1; Homo sapiens; ChIP-Seq
library_construction_protocol: BLaER1 cells were washed with PBS and fixed with 1% formaldehyde for 10 min at RT before quenching the reaction with 0.6 M glycine. Fixed cells were washed with cold PBS and lysed in 50mM TRIS pH 8, 10mM EDTA and 1% SDS. Lysates were sonicated using the Bioruptor (Diagenode) with cycles of 30s ON / 30s OFF in the high-power mode until a fragment length of 200-500bp was achieved. Lysates were mixed with dilution buffer to a final concentration of 20 mM TRIS pH 8, 150 mM NaCl, 2 mM EDTA ,1% Triton-X100 and 0.1% SDS. 2.5% drosophila melanogaster S2 cell derived chromatin (prepared similarly) was spiked into the samples. Lysates were pre-incubated at 4°C for 2 h with ProteinA-coated magnetic beads (G&E) previously blocked in TE with 1% BSA. After removal of the beads, target and spike-in antibodies were added to the lysates and incubated at 4°C overnight. Fresh blocked beads were added and incubated with the lysates for 2h at 4°C. Beads were washed with RIPA buffer (150 mM NaCl, 50 mM TRIS pH 8, 0.1% SDS, 0.5% Na-deoxycholate, 1% Igepal), high salt buffer (500 mM NaCl, 50 mM TRIS pH 8, 0.1% SDS, 1% Igepal), LiCl buffer (250 mM LiCl, 50 mM TRIS pH 8, 0.5% Na-deoxycholate, 1% Igepal) and TE wash buffer (10 mM TRIS-HCl, 1 mM EDTA, 50 mM NaCl) and protein DNA complexes were eluted with 100mM NaHCO3 + 1% SDS. ChIP and input samples were incubated overnight at 55°C with 200mM NaCl, 10mM EDTA, 20mM Tris-HCl pH 6.5 and 300 µg/ml Proteinase K (Thermo Fisher). DNA was isolated using the ChIP DNA Clean & Concentrator kit (Zymo Research). NEBNext Ultra II liabrary prep was performed accoring to manufacturer's instructions