title: GSM5840564: PRO_25HD_5min_rep1; Homo sapiens; OTHER
library_construction_protocol: Precision Run-on sequencing (PRO-seq) experiments were performed as previously described (Mahat, D. B. et al, Nat Protoc 11, 1455-1476, 2016) with modification. Briefly, for nuclei isolation, RUES1 cells were incubated with swelling buffer (10 mM Tris-HCl pH7.5, 2 mM MgCl2, 3 mM CaCl2) for 5min on ice and then incubated with lysis buffer (swelling buffer with 0.5% NP-40 and 10% glycerol) for 5 min on ice, before being re-suspended in 100μl of freezing buffer (50 mM Tris-Cl pH8.0, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA). For Run-on assay, an equal volume of reaction buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 300 mM KCl, 1 mM DTT, 20 units of SuperaseIn, 1% sarkosyl, 500 μM ATP, GTP, bio-UTP and bio-CTP) was added into each sample before incubation at 30°C for 5 min. The nuclear run-on RNA was then extracted with TRIzol LS reagent (Invitrogen) and subjected to hydrolysis, buffer exchange and purification by streptavidin beads (Thermo Fisher). 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. PRO-seq