GSM8193511: WT_DIF_H2AK119Ub_Rep2; Homo sapiens; ChIP-Seq
Biological duplicates of SH-SY5Y cells were grown in two 15 cm dishes per condition, which were processed independently. SH-SY5Y cells were incubated in differentiation medium with or without USP7 inhibitor FT671 (10 M) for 3 days, after which cells were harvested by trypsinization, followed by cell counting. For crosslinking, 20 million cells were resuspended in 10 ml PBS. Cells were fixed on a roller mixer by addition of 1% formaldehyde for 10 minutes. Crosslinking  was quenched by the addition of 125 mM glycine for 5 minutes.  Next, cells were washed twice with PBS and lysed in 1.8 ml lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS supplemented with aprotinin (1g/ml), AEBSF (0.2 mM), leupeptin (1 g/ml) and pepstatin A (1g/ml)). These protease inhibitors were present in all subsequent steps until elution. Cross-linked chromatin was fragmented to approximately 200 base pair (bp) DNA length in a Bioruptor® Standard sonication system. The normalization reference chromatin was prepared from Drosophila S2 cells. Approximately 80 million cells (one 15 cm dish) were harvested and subsequently fixed in 12.5 ml PBS with 1% formaldehyde for 10 minutes as above. After two wash steps with PBS, cells were lysed in 1.2 ml 50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 0.75% SDS supplemented with protease inhibitors. The Drosophila chromatin was sheared to approximately 200 bp DNA length in a Bioruptor® Pico sonication system. SH-SY5Y chromatin (185 µl) was mixed with S2 chromatin (15 µl), diluted 10 fold in dilution buffer (20 mM Tris, pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl and 1% Triton X-100) and cleared by centrifugation at 16000 g for 25 minutes. For BCOR chIP, double amounts of chromatin (370 µl of SH-SY5Y chromatin and 30 µl of S2 chromatin) were used, divided over two tubes. The supernatant was transferred to a new 2 ml tube and incubated overnight at 4 °C with either 5 µl anti-H2AK119ub1 (D27C4, Cell Signaling Technology #8240), 15 µl anti-H3K27me3  (C36B11, Cell Signaling Technology, #9733) or 10 µl anti-BCOR (E6V3R, Cell Signaling Technology, #63972). Next morning, 50 µl of magnetic Dynabeads™ Protein A slurry, which was blocked overnight with 1% BSA in dilution buffer, was added, and incubation was continued for an additional 3.5 hour. Next, beads were washed five times with 20 mM Tris, pH 8.0, 2 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, followed by a final wash step with 20 mM Tris, pH 8.0, 2 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100 and 500 mM NaCl. DNA was eluted with elution buffer (0.1 M NaHCO3, 1% SDS, 0.5 mg/ml proteinase K) for 2 hours at 37 °C, followed by overnight incubation at 65 °C. The eluted DNA was subsequently purified by phenol/chloroform extraction (2 times), followed by ethanol precipitation. ChIP-Rx-seq libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, #E7645L) according to the instructions of the manufacturer. AMPure® XP Beads (Beckman Coultier) were used for cleanup. Sequencing was performed by BGI Tech Solutions (Hong Kong) Co., Ltd. on an DNBSEQ-G400 sequencing platform.