GSM5770685: GFP-USP7-H2AK119Ub-Rep1; Homo sapiens; ChIP-Seq
Biological duplicates of DLD1-KO-Dox-USP7, HAP1 wt and HAP1 RING1A/B-KO cells were grown in two 15 cm dishes per condition. GFP-USP7 expression in DLD1-KO-Dox-USP7 cells was induced by addition of doxycycline  (1 g/ml) for three days (+Dox). Two other plates were mock treated (-Dox). Cells were harvested by trypsinization, followed by cell counting. For crosslinking, 17 million cells were suspended in 10 ml PBS. Cells were fixed by the addition of 1% formaldehyde for 10 minutes on a roller mixer. Fixation was stopped by the addition of glycine (125 mM). Next, cells were washed twice with PBS and lysed in 1 ml 1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0, supplemented with the following protease inhibitors. Cross-linked chromatin was sheared to approximately 200 bp of DNA length in a Bioruptor® Pico sonication system. The normalization reference exogenous genome was generated from 80 million Drosophila S2 cells (one 15 cm dish), which were harvested by “up-and-down” pipetting. Fixation was performed as above with 1% formaldehyde in PBS for 10 minutes. S2 cells were lysed in 1.2 ml  0.75 % SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0, supplemented with protease inhibitors. Drosophila chromatin was sheared to 200-300 bp of DNA length. DLD1-KO-Dox-USP7 chromatin (180 µl) was combined with 20 µl of S2 chromatin and diluted 10-fold with dilution buffer (1% Triton X-100, 2mM EDTA, 150 mM NaCl and 20 mM Tris pH 8.0), cleared by centrifugation at 15,000 g for 20 min. The supernatant was transferred to a fresh tube and incubated overnight at 4 °C in the presence of either 10 µl anti-Tri-Methyl-Histone H3 (Lys27) (Cell Signaling Technology #9733) or 5 µl anti-Ubiquityl-Histone H2A (Lys119) (Cell Signaling Technology #8240) on a rotating wheel. Next morning, 50 µl of Dynabeads™ protein A slurry (Invitrogen), which was blocked overnight with 1 % BSA in dilution buffer was added and followed by a 3 hour incubation on a rotating wheel. Next, beads were washed 4x with wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0) containing 150 mM NaCl and 1x with wash buffer containing 500 mM NaCl. DNA was eluted with 0.1 M NaHCO3, 1% SDS and 0.5 mg/ml proteinase K for two 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. KAPA Hifi kit according to manufacturer's instruction ChIP-Rx-seq