GSM9168771: Pro_WT_rep3; Mus musculus; OTHER
5 million nuclei were collected by sucrose gradient centrifugation. Nuclear run-on was performed at 37 °C for 5 min by mixing nuclei with 2 X run-on master mix (10 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 1 mM DTT, 300 mM KCl, 50 uM Biotin-11-CTP, 0.5 uM CTP, 250 uM each ATP/CTP/UTP, 10 U RNase inhibitor, 1% sarkosyl), followed by Trizol total RNA extraction. RNA was hydrolyzed with 0.2 N NaOH on ice for 12 min, neutralized with 0.55 M Tris-HCl pH 6.8, and buffer-exchanged with Bio-Rad P30 columns. Biotin-labeled RNA was enriched using Dynabeads MyOne Streptavidin C1 beads, treated with RppH (NEB) and T4 PNK (NEB), and sequentially ligated to 5' and 3' RNA adaptors with Streptavidin bead enrichments after each ligation. Reverse transcription was followed by PCR with indexed Illumina adaptors (8 cycles). 200-500 bp products were selected by SPRIselect beads (Beckman Coulter) to remove adaptor dimers. Libraries were then amplified to full scale with cycle number determined by test PCR, SPRIselect beads-purified, and sequenced via Illumina paired-end 150 bp sequencing.