GSM9137007: CHIP031, Input, siControl, Rep2; Homo sapiens; ChIP-Seq
ChIP-seq was performed as previously described(Shah et al. 2023; 2021). HCT116 cells were crosslinked in culture by addition of methanol-free formaldehyde (Pierce, final 1% v/v) for 10minutesat room temperature with gentle rotation. Crosslinking was quenched by addition of glycine (final 125 mM) for 5minuteswith gentle rotation. Media was discarded and replaced with PBS; cells were scraped and transferred to conical tubes and pelleted by centrifugation (1500 rpm, 3 minutes at room temperature). Resulting pellets were flash frozen in liquid nitrogen and stored at −80°C. For ChIP, 30μL protein G magnetic beads (per ChIP sample; ThermoFisher) were washed 3 times in blocking buffer (0.5% BSA in PBS); beads were resuspended in 250μL blocking buffer and 2μg antibody (Lamin B1, Abcam, ab16048; H3K9me2, Abcam, ab1220; H3K9me3, Abcam, ab8898; H3K27me3, Cell Signaling Technology, #9733) and rotated at 4°C for at least 6 hours. Crude nuclei were isolated from frozen crosslinked cells as follows: cell pellet (from 10cm plate or equivalent cell number) was resuspended in 10mL cold Lysis Buffer 1 (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, and protease inhibitors), and rotated at 4°C for 10 minutes, followed by centrifugation (1500 rpm at 4°C for 3 minutes). Supernatant was discarded and the pellet was resuspended in 10mL cold Lysis Buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, and protease inhibitors), and rotated at room temperature for 10 minutes, followed by centrifugation (1500 rpm at 4°C for 3 minutes). Supernatant was discarded and nuclei were resuspended/lysed in 1mL cold Lysis Buffer 3 (10mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, and protease inhibitors) and transferred to pre-chilled 1mL Covaris AFA tubes (Covaris). Samples were sonicated using a Covaris S220 sonicator (High Cell Chromatin Shearing: PIP 140W, CPB 200, Duty Factor 5%, 15 minutes). Lysates were transferred to tubes and Triton X-100 was added (final 1%) followed by centrifugation (top speed, 10 minutes at 4°C in microcentrifuge). Supernatant was transferred to a new tube; protein concentration was measured by Bio-Rad Protein Assay Kit (Bio-Rad, Protein Assay Kit #5000002). Antibody-conjugated beads were washed 3 times in blocking buffer, resuspended in 50μL blocking buffer and added to 500μg input protein for overnight incubation with rotation at 4°C. 50μg lysate was stored at −20°C for input. On day 2, beads were washed 5 times in 1mL RIPA buffer (50mM HEPES-KOH pH 7.5, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) with inversion to mix followed by 2-minute incubation on ice for each wash. Beads were washed in 1mL final wash buffer (1xTE, 50mM NaCl) for 2 minutes at room temperature before final resuspension in 210μL elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS). To elute, beads were incubated with agitation at 65°C for 30 minutes. 200μL eluate was removed to a fresh tube, and all samples (ChIP and reserved inputs) were reverse-crosslinked overnight at 65°C with agitation for a minimum of 12 hours, but not more than 18 hours. 200μL 1xTE was added to reverse crosslinked DNA to dilute SDS, and samples were treated with RNaseA (final 0.2mg/mL RNase; 37°C for 2 hours) and Proteinase K (final 0.2mg/mL Proteinase K; 55°C for 2 hours) before phenol:chloroform extraction and resuspension in 10mM Tris-HCl pH 8.0. ChIP and input DNA were quantified by Qubit (ThermoFisher) before library preparation using the NEBNext Ultra II DNA library prep kit according to manufacturer's instructions (NEB). Samples were indexed for multiplex sequencing with dual unique indices (NEBNext Multiplex Oligos for Illumina). Library quality was analyzed by BioAnalyzer (Agilent Genomics) and quantified using qPCR (NEBNext Library Quant Kit). Libraries were pooled for multiplex sequencing, re-quantified, and sequenced on the Illumina NextSeq1000 platform (121bp single-end sequencing).