title: GSM2936611: BJ.C.gro.rep1.lane2; Homo sapiens; OTHER
library_construction_protocol: 5 × 106 nuclei 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, 1 mM 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 total RNA was extracted with Trizol LS (Invitrogen) according to the manufacturer's instructions. RNA was fragmented using fragmentation reagents (Ambion) and the reaction was purified through p-30 RNasefree spin column (BioRad). BrU-labeled RNA was immunoprecipitated with anti-BrdU agarose beads (Santa Cruz), washed one time in binding buffer, one time in low salt buffer (0.2× SSPE, 1 mM EDTA, 0.05% Tween-20), one time high-salt buffer (0.25× SSPE, 1 mM EDTA, 0.05% Tween-20, 137.5 mM NaCl) and two times in TET buffer (TE with 0.05% Tween-20). 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 isolated with Trizol LS. After the binding step, BrU-labeled RNA was treated with tobacco acid pyrophosphatase (TAP, Epicenter) to remove 5′-methyl guanosine cap, followed by T4 polynucleotide kinase (PNK; NEB) to remove 3′-phosphate group. BrU-containing RNA was 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 libraries were prepared using TruSeq Small RNA kit (Illumina) following manufacturer's 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 amplified by PCR for 12 cycles. Finally, amplicons were cleaned and size-selected using Agencourt AMPure XP (Beckman Coulter), quantified in a Bioanalyzer 2100 (Agilent), and sequenced in a HiSeq 2500 (Illumina) GRO-seq
attributes.tag: GEO Accession
attributes.value: GSM2936611