GSM2492641: FAIRE-seq, EpRas, siEtv4/5 set 1, TGF-beta treated, 4 days; Mus musculus; FAIRE-seq
FAIRE samples were obtained based on a protocol as described previously (Giresi and Lieb, 2009; Waki et al., 2011). In brief, cells were fixed with final 1% formaldehyde for 5 min at room temperature and stopped the fixation by adding 2.5 M glycine (final 125 mM) for 5 min at room temperature. After washing fixed cells with cold phosphate-buffered saline (PBS) twice, cells were scraped with 1 ml cold PBS and centrifuged. Collected cells were lysed in 800 ul of MC lysis buffer (10 mM NaCl, 10 mM Tris-HCl pH 7.5, 3 mM MgCl2, 0.5% NP-40) and incubated on ice for 10 min. After centrifuged at 8,000 rpm at 4°C for 4 min, the pellet of cells was re-suspended in 400 ul of sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10 mM EDTA, protease inhibitor [P8304; Sigma-Aldrich]) and incubated on ice for 10 min. Next, DNAs in cell lysate were sonicated 15 cycles for 30 sec each time at intervals of 30 sec with Bioruptor UCW-201 (Cosmo Bio). After centrifuged at 14,000 rpm 4°C for 10 min, we added 200 ul of ChIP dilution buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100 Complete EDTA-free protease inhibitors (Roche)). Samples were centrifuged at 8,000 rpm at 4°C for 4 min. After removal of a control aliquot, supernatants were purified by phenol/chloroform extraction and reverse-crosslinked by overnight incubation at 65°C. Genomic DNA was purified by ethanol precipitation and extracted by RNase-free water after elution with a PCR purification kit (Qiagen). Libraries were made using Ion Fragment Library Kit (Thermo Fisher Scientific) and sequenced with Ion Proton (Thermo Fisher Scientific)