title: GSM3560401: RNF2-RNF2KO; Mus musculus; ChIP-Seq
library_construction_protocol: ES cells were treated with 100 nM 4-OHT and 10 μM emricasan (Cayman Chemical) for 4 days. For RNF2 ChIP, nuclear extracts were prepared from 2×10^7 cells using the truChIP High Cell Chromatin Shearing Kit (Covaris). Chromatin was sheared to 200-700 bp with a Covaris E220 sonicator (settings: duty cycle 5%, peak incident power 140 W, cycles per burst 200, processing time 12 min, temperature 4oC, degassing mode continuous, volume 1.0 ml in TC12x12 AFA tubes). Bap1C91A/- and Bap1-FLAG tag cells were crosslinked in 2 mM disuccinimidyl glutarate (Thermo Fisher Scientific) at room temperature for 30 min prior to nuclear extracts following the process above. Sheered nuclear extracts were centrifuged at 16000xg for 5 min, and then the soluble fractions were supplemented with 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20mM Tris-HCl pH 8.1 and 1× protease inhibitor cocktail (Sigma). Next, anti-RNF2 (Abcamab3832), anti-BAP1 (Genentech, PRO424270), or anti-FLAG (M8823 Millipore) antibodies were added that had been pre-coupled to protein A-Dynabeads (Life Technologies). Libraries were prepared using standard protocols for the Ovation Ultralow System. Briefly, samples were PCR-amplified and sized using the Agilent 2100 Bioanalyzer. Barcoded libraries were sequenced 50 bp single end on HiSeq 2500 (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM3560401