GSM9271332: Parental U2OS cells, Control, Rep 3; Homo sapiens; OTHER
Cells were resuspended in cytoplasmic lysis buffer (0.15% NP-40, 10 mM Tris-HCl pH 7.0, 150 mM NaCl) before being incubated with ice for 5 min. The lysate was then poured on top of Sucrose buffer (10 mM Tris-HCl pH 7.0, 150 mM NaCl, 25% sucrose) and centrifuged at 16,000g for 10 min. The nuclei were resuspended in nuclei wash buffer (0.1% Triton X-100, 1 mM EDTA, in 1x PBS) before being centrifuged at 1500g for 1 min. The pellet was resuspended in glycerol buffer (20 mM Tris-HCl pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol, 0.85 mM DTT), and then 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 before the sample was vortexed and put on ice. The sample was centrifuged further for 2 min at 18,500g, and the nucleoplasm fraction was extracted by removing the supernatant. The chromatin was resuspended in chromatin resuspension buffer (25 μM α-amanitin, 50 Units SUPERaseIN, Protease inhibitors cOmplete, in 1x PBS). The chromatin fraction was extracted using the Zymol RNA extraction kit following manufacturer's instructions. RNA quality and concentration was determined using the 4200 tapestation system. NEBNext® Ultra™ II RNA Library Prep Kit for Illumina