GRO-seq

Cells 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 7 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), and 1% sarkosyl. Nuclear RNA was isolated with Trizol (Invitrogen). DNAse treatment was performed with Turbo DNA-free kit (Invitrogen). RNA was purified with Micro Bio-Spin P-30 Gel Columns (Bio-Rad), fragmented with RNA Fragmentation Kit (Invitrogen), and treated with 10 units RppH (NEB) and 30 units T4 PNK (NEB). RNA immunoprecipitation was performed with anti-BrdU-conjugated agarose beads (Santa Cruz Biotechnologies). Library preparation was performed with TruSeq Small RNA Library Preparation Kit (Illumina). GRO-seq reads were aligned with HISTAT2 software to Ad5 and human (hg19) genomes and normalized to the number of reads aligned to hg19. Pause indexes (TSS to +200 counts)/(+201 to TTS counts) were calculated using HTSeq software. Genes determined to be affected by A-485 underwent additional profiling using Homer software (http://homer.ucsd.edu/homer/; Heinz et al., 2010) for biological processes gene ontology enrichment as well as TF motif analysis in promoter regions (± 300 bp from TSSs).