GSM5944508: svtRNA1-2 KO rep1; Homo sapiens; OTHER
GSM5944508
Cells were harvested from 15 cm dishes and lysed in HLB+N buffer (10mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40), and underlaid with HLB+NS buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 10% sucrose), before centrifugation at 420g for 5 minutes. Nuclear pellets were then resuspended in NUN1 buffer (20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, 50% glycerol). Chromatin was extracted by addition of NUN2 buffer (20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% NP-40, 1M urea) on ice for 15 minutes with vortexing. Samples were centrifuged at 17,000g for 10 minutes and chromatin pellets collected. Chromatin was digested by the addition of Proteinase K and Turbo DNase (Thermo Fisher). RNA was extracted from the chromatin samples using TRIzol (Invitrogen) and chloroform extraction, followed by the Monarch® Total RNA Miniprep Kit (NEB) according to the manufacturer's instructions and eluted in water. RNA libraries were prepared for sequencing using standard Illumina protocols