GSM3536993: sgRNA1830 targeting ERa binding site and serves here as another control; Homo sapiens; OTHER
GRO-Seq was performed as described before with minor modifications. Briefly, 5x106 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, 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 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 RNAse-free spin column (BioRad). BrU-labeled RNA was immunoprecipitated with anti-BrU agarose beads (Santa Cruz), washed one time in binding buffer, one time in low salt buffer (0.2X SSPE, 1 mM EDTA, 0.05% Tween-20), one-time high salt buffer (0.25X 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-labbeled 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)
GEO
GSM3536993
GEO Accession
GSM3536993