title: GSM6724043: H3K27me3-RingKO-+indRing1a-WT-DOX-Rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: Biological duplicates of DLD1 WT, DLD1-DKO-Dox-RING1A, DLD1-KO-Dox-USP7, HAP1 WT, and HAP1 RING1A/B-KO cells were grown in two 15-cm dishes per condition, which were processed independently. GFP-USP7 or GFP-RING1A expression was induced by addition of Dox (1 μg/ml) for 3 days (+Dox). Two other plates were mock-treated (−Dox). Cells were harvested by trypsinization, followed by cell counting. For cross-linking, 17 million cells were suspended in 10 ml of PBS. Cells were fixed by the addition of 1% formaldehyde and incubation on a roller mixer for 10 min. Fixation was stopped by the addition of glycine (125 mM). Next, cells were washed twice with PBS and lysed in 1 ml of lysis buffer: 50 mM tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS, supplemented with protease inhibitors pepstatin A (1 μl/ml), leupeptin (1 μg/ml), aprotinin (1 μg/ml), and 0.2 mM AEBSF. These protease inhibitors were present in all subsequent buffers until elution. Cross-linked chromatin was sheared to approximately 200–base pair (bp) DNA length in a Bioruptor Pico sonication system. The normalization reference exogenous genome was isolated from ~80 million Drosophila S2 cells (one 15-cm2 dish). Fixation was performed as above with 1% formaldehyde in PBS for 10 min. S2 cells were lysed in 1.2 ml of 0.75% SDS, 10 mM EDTA, and 50 mM tris-HCl (pH 8.0), supplemented with protease inhibitors. Drosophila chromatin was sheared to 200- to 300-bp DNA length. DLD1-KO-Dox-USP7 chromatin (180 μl) was combined with 20 μl of S2 chromatin and diluted 10-fold with dilution buffer [20 mM tris (pH 8.0), 1% Triton X-100, 2 mM EDTA, and 150 mM NaCl], cleared by centrifugation at 15,000g for 20 min. The supernatant was transferred to a fresh tube and incubated overnight at 4°C in the presence of either 10 μl of anti-H3K27me3 (Cell Signaling Technology, #9733) or 5 μl of anti-H2AK119ub1 (Cell Signaling Technology, #8240) on a rotating wheel. The next morning, 50 μl of Dynabeads protein A slurry (Invitrogen), which was blocked overnight with 1% BSA in dilution buffer, was added, followed by a 3-hour incubation. Next, beads were washed four times with wash buffer [20 mM tris-HCl (pH 8.0), 2 mM EDTA, 0.1% SDS, and 1% Triton X-100] containing 150 mM NaCl, followed once with wash buffer containing 500 mM NaCl. DNA was eluted with 0.1 M NaHCO3, 1% SDS, and proteinase K (0.5 mg/ml) for 2 hours at 37°C, followed by an overnight incubation at 65°C. DNA was subsequently purified by double phenol/chloroform extraction, followed by ethanol precipitation. ChIP-Rx-seq libraries were prepared using the ThruPLEX DNA-Seq Kit (Takara Bio, R400675), according to the manufacturer's instructions. ChIP-Rx DNA was repaired to obtain blunt-ended double-stranded DNA followed by ligation of stem-loop adapters. The stem loop was cleaved off, and a library amplification was performed that extended the template with dual indices and sequence needed for posterior sequencing on an Illumina HiSeq 2500 sequencer (dual-indexed 50-bp paired-end reads).