GSM6926919: Scr2-RAD21; Homo sapiens; ChIP-Seq
ChIP-seq: ChIP was performed based on (Shah et al., Dev Cell 2021). HCT-116 cells were cross-linked in culture medium by addition of methanol-free formaldehyde (ThermoFisher, final 1% v/v) and incubated at room temperature for 10 minutes with gentle nutation. Crosslinking was quenched by addition of glycine (final 125 mM) and incubated at room temperature for 5 minutes with gentle nutation. Media was aspirated and replaced with cold DPBS. Cells were scraped and transferred to conical tubes, then pelleted by centrifugation (1500 rpm, 3 minutes, room temperature). Pellets were flash frozen on liquid nitrogen and stored at -80C.  For ChIP, 30 uL protein G magnetic beads (per sample; ThermoFisher) were washed three times in blocking solution (0.5% BSA in DPBS). Beads were then resuspended in 250 uL blocking solution and 2 ug antibody (RAD21, Abcam ab992; CTCF, Cell Signaling Technology 3418) was added. Beads and antibody were rotated at 4C for at least six hours. Nuclei were isolated from frozen cell pellets as follows: pellet was resuspended in 10 mL 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 4C for 10 minutes, followed by centrifugation (1500 rpm, 3 minutes, 4C). Supernatant was aspirated and the pellet was resuspended in 10 mL 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, 3 minutes, 4C). Supernatant was discarded and nuclei were resuspended in 1 mL 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). Nuclei were sonicated using a Covaris S220 sonicator (high cell chromatin shearing for 15 minutes; Covaris). Sonicated chromatin was transferred to 1.5mL microcentrifuge tubes and Triton-X 100 was added (1% final v/v) followed by centrifugation (top speed, 10 minutes, 4C). Supernatant was transferred to a new tube. Antibody-conjugated protein G beads were washed three times in blocking solution, resuspended in 50 ul blocking buffer, and added to 500 ug sonicated chromatin. Chromatin was rotated overnight at 4C. 50 ug lysate was reserved in a separate tube at -20C for input.   On day 2, beads were washed five times in 1 mL RIPA buffer (50mM HEPES-KOH pH 7.5, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate). Beads were then washed in 1 mL final wash buffer (1xTE, 50mM NaCl) for 2 minutes. Beads were finally resuspended in 210 uL elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS), and chromatin was eluted from beads by agitation at 65 °C for 30 minutes. 200 uL eluate was transferred to a new tube, and all samples (ChIP and input) were reverse cross-linked overnight at 65 °C with agitation for between 12 and 18 hours. 200 uL 1xTE was added to all samples, and samples were treated with RNase A (final 0.2mg/mL RNase; 37 °C for 2 hours) and proteinase K (final 0.2mg/mL Proteinase K; 55C for 2 hours). DNA was purified using phenol:chloroform extraction and resuspension in 10mM Tris-HCl pH 8.0.  In situ Hi-C: Hi-C libraries were generated from 10e6 cells using the Arima-HiC+ kit (Arima Genomics) according to manufacturer's instructions ChIP-seq: ChIP and input DNA were quantified by Qubit (ThermoFisher) before library preparation using the NEBNext Ultra II DNA library prep kit (NEB). Samples were indexed for either single or dual-index sequencing. Library quality was assessed on Bioanalyzer (Agilent) and quantified by qPCR (Kapa Biosystems).   In situ Hi-C: Libraries were prepared using the Kapa Hyper Prep Kit with KAPA Library Amplification Primer Mix (KK8502), according to manufacturer's recommendations. Libraries were validated for quality and size distribution using the Qubit dsDNA HS Assay Kit (Invitrogen, cat# Q32851), KAPA Library Quantification kit (Roche, Cat# KK4824) and TapeStation 2200 (Agilent). ChIP-seq: Quantified libraries were sequenced via Illumina sequencing using the NextSeq500 (single end, 75bp) and NovaSeq6000 (paired-end, 100bp) platforms. Data from the same libraries were merged for final analysis.  In situ Hi-C: Paired-end sequenced (61bp+61bp) on a NovaSeq 6000 (Illumina).