GSM8702297: [GRO-Seq] MCF10A-vec-0hr-3 (replica 3); Homo sapiens; OTHER
For GRO-seq: nuclei were isolated from tissue culture cells, counted the number, washed and frozen (1 x 10^6 of nuclei in 10 ul of freezing buffer) at -80C until ready for in vitro Nuclear Run-on (NRO) transcription reaction. When the Gro-seq preparation was ready, 100 ul of nuclei stock were mixed with 100 ul of 2x NRO buffer (containing, ATP, GTP, BrUTP and CTP) and incubated at 37C for 7 min. The reaction was stopped by adding TRI-LS reagent (Sigma) and purified RNA in 20 ul of water with RNase inhibitor (1U/ul). The collected RNAs were further treated with DNase I (Turbo DNA-free kit, Bio-Rad), RNA fragmentation by RNA fragmentation kit (Ambion), purified by Micro-Bio-Spin P-30, incubated with T4 polynucletide kinase (PNK) at 37C for 60 min, and inactivated the PNK at 75C for 5 min. The final Br-UTP incorporated RNAs were immunoprecipitated with beads (anti-BrdU antibody conjugated agarose; Santa Cruz; sc-32323 AC), washed beads, and eluted by 100 ul of 0.25M EDTA-TE for 4 times. The eluted RNAs were purified by EtOH precipitation, and the final RNAs were dissolved in 15 ul of water. For GRO-seq: performed by Takara SMATer smRNA library kit as manufactured suggested (polyadenylation, cDNA synthesis, PCR and clean up).