title: GSM1474238: ChrRNA_siXrn2; Homo sapiens; OTHER
library_construction_protocol: Chromatin RNA fraction was prepared from ~80% confluent HeLa cells in 100mm Dishes. Approximately 7x106 cells were washed with ice-cold PBS twice. The cells were lysed with ice-cold 4 ml of HLB/NP40 buffer (10 mM Tris-Hcl pH 7.5, 10 mM NaCl, 0.5% NP40 and 2.5 mM MgCl2) and incubated on ice for 5 min. After the incubation, 1 ml of ice-cold HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2 and 10 % Sucrose) was under-laid and then the nuclei were collected under 1,400 rpm centrifuge at 40C for 5 min. Isolated nuclei were resuspended in 1.25 ml of NUN1 solution (20 mM Tri-HCl pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete (Roche)) and added 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40, proteinase inhibitor 1xComplete and phosphatase inhibitor 1xPhosStop (Roche)). 15 min incubation was carried out on ice with mixing by max speed vortex for 5 sec every ~4 min and then chromatin pellets were precipitated under 13,000 rpm centrifuge at 4oC for 10min. Chromatin pellet was resuspended in 200 ml HSB (10mM Tris-HCl pH 7.5, 500 mM NaCl and 10 mM MgCl2) with 0.25 U/ml TURBO DNase (Life technologies) at 37oC for 10 min and then treated with Proteinase K for 10 min. RNA was extracted by Trizol reagent (Life technologies). This extraction steps were repeated three times. In prior to RNA library preparations, rRNAs were depleted using Ribo-Zero rRNA removal kits (Epicentre) from 5 mg of Chromatin RNA. RNA was also fragmented 150-200 nt by heat treatment (94oC) for 15 min in 1xNEB first strand synthesis buffer. 100 ng or chromatin RNA was used for RNA library preparations. These were carried out according to NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB). Deep sequencing using Hiseq2000 and Hiseq2500 were performed by the Wellcome Trust Centre for Human Genetics (WTCHG) Oxford UK.