title: GSM7720585: HEK293T, WT, groseq, Rep1; Homo sapiens; OTHER
library_construction_protocol: 5x106 cells per nuclear run-on reaction were harvested and incubated in swelling buffer (10 µM Tris-HCl, 2 mM MgCl2, 3 mM CaCl2). Nuclei were isolated with lysis buffer (10 µM Tris-HCl, 2 mM MgCl2, 3 mM CaCl2, 10% glycerol, 1% NP-40). Nuclear run-on was performed at 30°C for 5 min in 10 mM Tris-HCl pH 8, 5 mM MgCl2, 300 mM KCl, 1 mM DTT, 500 µM ATP, 500 µM GTP, 500 µM Br-UTP, 2 µM CTP, 200 U/mL Superase In RNase Inhibitor (Invitrogen #AM2696), and 1% sarkosyl. Nuclear RNA was isolated with Trizol (Invitrogen #15596026). RNA was purified with Micro Bio-Spin P-30 Gel Columns (Bio-Rad # 7326250), fragmented with RNA Fragmentation Kit (Invitrogen # AM8740), and treated with 15 units mRNA decapping enzyme (NEB # M0608S) and 30 units T4 PNK (NEB # M0201S). RNA immunoprecipitation was performed 3 times with anti-BrdU-conjugated agarose beads (Santa Cruz Biotechnologies # sc-32323 AC). Library preparation was performed with TruSeq Small RNA Library Preparation Kit (Illumina # RS-200-0012 following the manufacturer's protocol.