title: GSM4156275: ChIP-seq of ARID2 in ARID1-less H2.35 cells [ARID1-less-ARID2-3]; Mus musculus; ChIP-Seq
library_construction_protocol: WT H2.35 cells expressing a Dox-inducible scramble shRNA and the indicated 3xTy1-tagged proteins were used for control ChIP. ARID1B knockout H2.35 cells expressing a Dox-inducible ARID1A shRNA and the indicated 3xTy1-tagged proteins were used for ARID1-less ChIP. Expression of exogenous proteins was titrated by transfecting different amount of pT3 plasmids. Transfections that resulting similar expression levels with their endogenous counterparts were used. To do ChIP, cells were induced with 1μg/ml Dox for 3 days and crosslinked by addition of 2mM disuccinimidyl glutarate (0.5M DMSO stock) for 45 min, followed by addition of 1% formaldehyde (37% stock) for 10 min. Crosslinking was quenched by addition of final 125 mM glycine solution (2.5 M stock) to the medium for 5 min. The cells were washed with PBS twice, scraped and collected by centrifugation. Cell pellets were resuspended in RIPA-0 (10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 0.25% sarkosyl, pH8.0) and sonicated until DNA to an average size of 200 bp. The sonicated lysates were cleared by centrifugation at 20,000g for 10 min and supplemented with NaCl to final concentration of 0.3 M. The lysates were then incubated with 5 μg Ty1 antibody overnight followed by incubation with 25 μl protein A/G Dynabeads for another 3 hours with rotation. After incubation, the beads were washed with 1 ml following buffers, twice with RIPA-0, twice with RIPA-0.3 (RIPA-0 supplemented with 0.3 M NaCl), twice with LiCl buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5% sodium deoxycholate, 0.5% NP-40, 250 mM LiCl, pH 8.0), and twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The DNA was eluted with 150 μl SDS elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) followed by incubation at 65°C overnight. The eluted DNA was treated with 2 μl RNase A (0.5 μg/μl) for 30 min at 37 °C followed by 2 μl Protease K (20 mg/ml) for 2 hours at 37 °C, and purified using MinElute PCR purification kit (Qiagen). The purified DNA was used for library preparation using NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina Kit according to manufacturer's protocol.