GSM1634454: Myc.gro.rep1; Homo sapiens; RNA-Seq
Cells were treated with Doxycyline for 5h and 5 x 106 nuclei per condition were isolated and incubated 5 min at 30°C with equal volume of reaction buffer for the nuclear-run on (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). 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). BrUTP labeled nascent transcripts were immunoprecipiated with anti-BrUTP 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, BrUTP-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. End-repair of the enriched BrUTP-incorporated RNAs were achieved by the treatment with tobacco acid pyrophosphatase (TAP; Epicenter) to remove 5’-methyl guanosine caps, and followed by T4 polynucleotide kinase treatment (PNK; NEB) to remove 3’-phosphate group at low pH. Then, the BrU-incorporated RNAs were treated with T4 PNK 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 generated from two biological replicates using TruSeq Small RNA kit from Illumina. Final libraries were two times cleaned-up and size-selected by Agencourt AMPure XP (Beckman Coulter)
GEO Accession
GSM1634454