title: GSM9443257: NBSD_Input; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP sequencing and CUT&RUN sequencing Histone ChIP-sequencing was performed as previously described17,24. 2 x 107 cells per sample were crosslinked in 1% formaldehyde for 10 minutes and quenched in 0.20M glycine for 5 minutes. Cells were lysed using an SDS buffer (100 mM NaCl, 50 mM Tris-Cl (pH 8.0), 5 mM EDTA (pH 8.0), 0.2% NaN3, 10% SDS) with 1X HALT protease inhibitor (Thermo cat#78429) and incubated at room temperature with gentle inversion for 2 minutes. Following centrifugation at 15,000 rpm nuclei were resuspended in IP buffer (two parts SDS buffer to one part triton dilution buffer (100 mM Tris-Cl (pH 8.0), 100 mM NaCl, 5 mM EDTA (pH 8.0) 0.2% NaN3, 5.0% Triton X-100)), and sheared for 15 minutes on a Covaris E220 ultrasonicator (5% duty cycle, peak power 140 W, cycles per burst 200) at 4 °C. Immunoprecipitation was performed with prepared antibody protein A magnetic beads conjugated overnight at 4°C with rotation. 10µg of antibody H2AK119ub (Cell Signaling Technology, 8240S) and 2 µL of the spike-in drosophila antibody (Active Motif, 61686) for normalization were added to each sample and rotated at 4°C overnight. The next day, chromatin were sequentially washed with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl), twice with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 500 mM NaCl), twice with LiCl wash buffer (0.25 M LiCl, 1% IGPAL-CH 630, 1% deoxycholic acid, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA), once with ice cold Tris-EDTA (TE), and then eluted in fresh elution buffer. IP samples were decrosslinked with 2.5 M NaCl, proteinase K, and RNAse A and then incubated at 65°C for 18 hours. DNA for all samples was isolated by incubating with AMPure beads (Beckman Coulter, A63880) for 20 minutes at room temperature, washing twice with 80% ethanol, and eluting in 1x TE. CUT&RUN sequencing was performed with the EpiCypher CUTANA ChIC/CUT&RUN (v5.0 #14-1048)  Kit according to manufacturer instructions. Briefly, ~1 x 10^6 cells were adsorbed to activated ConA beads and washed. 0.5 µg of the following antibodies were incubated into specified reaction tubes at 4°C overnight: RING1B (CST #5694), PCGF1 (Invitrogen #49390), PCGF1 (Abcam #259943), EZH2 (CST #5246), and isotype control supplemented with 0.01%  digitonin. The next morning cells were washed with cell permeabilization buffer and manufacturer recommended volume of pAG-MNase and calcium chloride added to each reaction. Chromatin was digested for 2 hours at 4°C before quenching. DNA was purified with AMPure XP beads prior to library preparation using the CUTANA CUT&RUN library Prep Kit (14-1001) according to manufacturer instructions.