GSM9449429: HeLa mock rep2; Homo sapiens; OTHER
Both infected and noninfected samples of HeLa cells (ATCC CCL-2) were prepared as two biological replicates. Cell nuclei were extracted post-infection at the indicated times. Cells were washed with PBS and scraped into swelling buffer containing 10 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 3 mM CaCl2, 1 U/ml SUPERase-In and 1 U/ml RNAsin plus in ultrapure water (Gibco 15564-027, Invitrogen AM9530G, Sigma-Aldrich 21115-100ml, Invitrogen 10977-035, Invitrogen AM2694, Promega N2611). Cells were centrifuged at 400 x g for 10 minutes. Formed cell pellet was resuspended into swelling buffer containing 10 % glycerol and 4 U/ml SUPERase-In (ACROS ORGANICS 327255000, Invitrogen AM2694). Nuclei were extracted by incubating the cells in swelling buffer supplemented 10 % glycerol and 1% Igepal (Igepal®CA-630 I8896-50ml) for 5 minutes. Lysis buffer composted of swelling buffer, 0.5 % Igepal, 10% glycerol, 1 U/ml SUPERase-In and 1 U/ml RNAsin plus was added to the extracted nuclei. Nuclei were washed once with lysis buffer. Isolated nuclei were resuspended into freezing buffer containing 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 50 mM Tris-HCl (pH 8.3) and 2 U/ml SUPERase-In (Invitrogen AM9260G, Gibco 15564-027 Invitrogen 15568-025) and frozen. Nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. RNA was extracted with Trizol, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads and precipitated overnight. Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified using ChIP DNA Clean & Concentrator kit. The recovered cDNA was RNAseH treated, circularized, linearized, amplified for 11-14 cycles. The final product was ran on 10% TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit. Libraries were sequenced for 50 cycles on an Illumina HiSeq 2000 according to the manufacturer's instructions.