GSM2480871: siLuc (rep 2); Homo sapiens; RNA-Seq
Approximately 3x106 cells were resuspended in 500 µL of ice-cold RLB buffer, (10 mM Tris-Hcl, pH 7.5, 140 mM NaCl, 0.5% NP40, 1.5 mM MgCl2), and lysed by adding an equal volume of RLB buffer with 24% (m/v)  sucrose. Nuclei were collected by centrifugation (14,000 x g) at 4°C for 10 min. Isolated nuclei were resuspended in 120 µl of NUN1 buffer, (20 mM Tri-HCl, pH 7.9, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol, 0.125 mM PMSF, 1mM DTT), followed by addition of 1.2 ml NUN2 buffer, (20 mM HEPES-KOH pH 7.6, 7.5mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M urea, 1 % NP40, 1 mM DTT). Nuclei were incubated for 15 minute on ice with mixing by vortexing for 5 seconds every 5 min. Chromatin pellets were precipitated by centrifugation (14,000 x g) at 4°C for 10 min and then resuspended in 200 ml HSB buffer, (10 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM MgCl2), in presence of  4 U TURBO DNase (Ambion) and incubated at 37°C for 20 minutes. Followed a 20 minutes proteinase K treatment (Roche) at 37°C, chromatin RNA (chrRNA) was phenol-chloroform extracted and ethanol precipitated. RNA was resuspended in 200 µL of 1x TURBO DNase buffer in presence of 4 U TURBO DNase and incubated at 37°C for 30 minutes. The entire procedure of extraction, precipitation and DNase treatment was repeated twice. RNA was then extracted and precipitated again, prior to solubilisation in 75 µL of RNase free water.   Prior to RNA libraries preparation, rRNAs were depleted using Ribo-Zero Magnetic kit (Illumina) from 2.5 µg of ChrRNA. Libraries were prepared starting from chromatin RNA using the NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB) following the manufacturer’s instructions
GEO
GSM2480871
GEO Accession
GSM2480871