GSM4015603
GSM4015603: siTop1_Veh_1h_exp1; Homo sapiens; OTHER
The resultant nuclei were washed two more times with 10ml lysis buffer and finally resuspended in 100 μl of freezing buffer (50mM Tris-HCl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For the run-on assay, resuspended nuclei were mixed with an equal volume of reaction buffer (10mM Tris-HCl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of SUPERase-In, 1% sarkosyl, 250 μM A/GTP, 50 µM biotin-11-C/UTP (Perkin-Elmer) and incubated for 5 min at 30°C. The resultant nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol® LS reagent (Life Technologies, Cat# 10296-028) following manufacturer's instructions. NRO-RNA was fragmented to ∼200-500nt by alkaline base hydrolysis on ice for 30 min and and neutralized by adding 1× volume of 1 M Tris-HCl pH 6.8, Excessive salt and residual NTPs were removed by using P-30 column (Bio-Rad, Cat# 732-6250), followed by treatment with DNase I (Promega Cat# M6101) and antarctic phosphatase (NEB Cat# M0289L). Fragmented nascent RNA was bound to 10 µl of MyOne Streptavidin C1 dynabeads (Invitrogen, Cat# 65001) following the manufacturer's instructions. The beads were washed twice in high salt (2 M NaCl, 50 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 0.5 mM EDTA), once in medium salt (1M NaCl, 5 mM Tris-HCl pH 7.5, 0.1% Triton X-100, 0.5 mM EDTA), and once in low salt (5 mM Tris-HCl pH 7.5, 0.1% Triton X-100). Bound RNA was extracted from the bead using Trizol (Invitrogen, Cat# 15596-018) in two consecutive extractions, and the RNA fractions were pooled, followed by ethanol precipitation and PRO-seq libraries were prepared with NEBNext® Small RNA Library Prep kit (NEB, Cat# E7330).
GEO
GSM4015603
GEO Accession
GSM4015603