GSM9553635: BJ-hTert cells, KRASG12V expression, 48h induction, Rep 1; Homo sapiens; RNA-Seq
Cell fractionation was performed as previously described in (PMID: 25910208) with modifications described below. All steps were conducted on ice or at 4°C and in the presence of 20 μM α-amanitin (Sigma, A2263), 25 μM RNA polymerase I inhibitor CX-5461 (Merck, 5.09265), 25 μM RNA polymerase III inhibitor ML-60218 (Merck, 557403), 50 Units SUPERaseIN (Life Technologies, AM2696) and Protease inhibitors cOmplete (Roche, 11873580001). Briefly, 5 × 106 cells for each condition were resuspended and incubated in 0.15% NP-40, 10 mM Tris-HCl pH 7.0, 150 mM NaCl for 5min, then layered onto a sucrose buffer (10 mM Tris-HCl pH 7.0, 150 mM NaCl, 25% sucrose) and centrifuged at 16,000 g for 10 min. The supernatant carefully removed, and the nuclei pellet was gently resuspended in nuclei wash buffer (0.1% Triton X-100, 1 mM EDTA, in 1x PBS) and centrifuged at 1500 g for 1 min. The supernatant was removed, and the pellet was gently resuspended in glycerol buffer (20 mM Tris-HCl pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol, 0.85 mM DTT) before an equal volume of nuclei lysis buffer (1% NP-40, 20 mM HEPES pH 7.5, 300 mM NaCl, 1M Urea, 0.2 mM EDTA, 1 mM DTT) was added. Samples were vortexed, incubated on ice for 2 min and centrifuged at 18,500 g for 2 min. The supernatant was removed, and the chromatin pellet was immediately incubated with TRI Reagent® (Zymo Research) and stored at -80°C for RNA extraction with Direct-zol RNA Miniprep Kit. Approximately, 200-300 ng of input RNA in each repeat were depleted of ribosomal RNA with NEBNext® rRNA Depletion Kit v2 according to manufacturer's guidelines. Chromatin RNA-Seq was performed on chromatin fractions. NEBNext Ultra II 8482 Directional RNA Library Prep kit for Illumina