title: GSM5840560: GRO_16HD_30min_rep1; Homo sapiens; OTHER
library_construction_protocol: GRO-Seq experiments were performed as previously reported (Oh, S. et al. Nature s41586-021-03577-1 ,2021) with modification. Briefly, ~10  million RUES1 cells were washed 3 times with cold PBS and then sequentially swelled in swelling buffer (10mM Tris-Cl pH7.5, 2mM MgCl2, 3mM CaCl2) for 5 min on ice, harvested, and lysed in lysis buffer (swelling buffer plus 0.5% NP-40 and 10% glycerol). The resultant nuclei were washed one more time with 10mL lysis buffer and finally re-suspended in 100uL of freezing buffer (50mM Tris-Cl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For the run-on assay, re-suspended nuclei were mixed with an equal volume of reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of SUPERase-IN, 1% sarkosyl, 500uM ATP, GTP, and Br-UTP, 2uM CTP) and incubated for 5 min at 30°C. The resultant nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent (Invitrogen) following manufacturer's instructions. NRO-RNA was fragmented to ~300-500nt by alkaline base hydrolysis on ice and followed by treatment with DNase I and Antarctic phosphatase. These fragmented Br-UTP labeled nascent RNA was then immunoprecipitated with an anti-BrdU agarose beads (Sc32323ac, Santa Cruz Biotechnology) in binding buffer (0.5XSSPE, 1mM EDTA, 0.05% tween) for 3 hrs at 4°C with rotation. Purified RNA was treated with PNK before being used for cDNA synthesis by using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® Kit (NEB). Obtained cDNA template was amplified by PCR using the LongAmp® Taq 2X Master Mix (NEB) for deep sequencing. Purified RNA was treated with PNK before being used for cDNA synthesis by using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® Kit (NEB). Obtained cDNA template was amplified by PCR using the LongAmp® Taq 2X Master Mix (NEB) for deep sequencing. GRO-Seq