GSM1422218: MCF7 GROseq nutlin 2; Homo sapiens; OTHER
GRO-Seq was performed as described before (Core, L.J., Waterfall, J.J., and Lis, J.T., Science 322, 1845-1848 (2008)) with minor modifications. Briefly, 5 million nuclei per condition were isolated and incubated 5 min at 30°C with equal volume of reaction buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 1mM DTT, 300 mM KCL, 20 units of SUPERase In, 1% sarkosyl, 500 µM ATP, GTP, and Br-UTP, 0.2 µM CTP+32P CTP) for the nuclear-run on.  The reaction was stopped and RNA extracted with Trizol LS (Invitrogen) according to the manufacturer’s instructions. Base hydrolysis was performed using RNA fragmentation Reagents (Ambion) and the reaction was purified through p-30 RNAse-free spin column (BioRad). BrU labeled nascent transcripts were immunoprecipitated with anti-BrU agarose beads (Santa Cruz Biotech), washed once in binding buffer, once in low salt buffer (0.2X SSPE, 1 mM EDTA, 0.05% Tween-20), once in high salt buffer (0.25X SSPE, 1 mM EDTA, 0.05% Tween-20, 137.5 mM NaCl) and twice in TET buffer (TE with 0.05% Tween-20). Then, BrU-incorporated RNA was eluted with elution buffer (20 mM DTT, 300 mM NaCl, 5 mM Tris-Cl pH 7.5, 1 mM EDTA and 0.1% SDS) and RNA isolated with Trizol LS.  After the binding step, the BrU-RNAs were treated with tobacco acid pyrophosphatase (TAP; Epicenter) to remove 5’-methyl guanosine caps, then with T4 polynucleotide kinase (PNK; NEB) to remove 3’-phosphate group at low pH. The BrU-RNAs were treated with T4 PNK again at high pH in the presence of ATP to add 5’-phosphate group. The reaction was stopped and RNA was extracted with Trizol LS. Sequencing library was prepared using TruSeq Small RNA kit (Illumina) following manufacturers instructions. Briefly, end-repaired RNA was ligated to RNA 3’ and 5’ adapters, followed by RT-PCR amplification. cDNA was purified using Agencourt AMPure XP (Beckman Coulter) and PCR amplified during 12 cycles. Final libraries were cleaned-up and size-selected by two times Agencourt AMPure XP and sequenced according to Illumina’s protocol in a HiSeq 2000 platform.