title: GRO-R4
design: Total RNA was extracted using Trizol (Invitrogen) and treated with DNase I (Thermo Scientific) according to manufacturers instructions. The immunoprecipitation of Br-UTP labeled RNA was performed using Dynabeads Protein G (Invitrogen) linked to Anti-BrdU antibody (Abcam). The magnetic beads preparation and the RNA immunoprecipitation were performed according to the manufacturers instructions. _x0004__x0005_RNA purification was performed with ethanol precipitation adapted from Gardini (2017): addition of 1 g/L glycogen, 300 mM NaCl and 1 mL of cold 100% ethanol followed by overnight incubation at -20 C_x0004_ _x0005_. Tubes were centrifuged (12000 x g, 30 min., 4 C) and washed three times with 75% ethanol during10 minutes incubation (room temperature) to dissolve salts. Then, the pellet was air-dried, resuspended in 5 L of nuclease-free water and incubated for 15 minutes (room temperature). The quantification of samples was performed with NanoDrop 2000 Spectrophotometer and integrity of RNA was evaluated through electrophoresis in 1.5% agarose gel. RNA fragments (biological triplicates and a background sample) were deep sequenced using smRNA-Seq Kit for Illumina (1 x 75 bp) at Polyomics Facility (Glasgow University), with no poly A selection or rRNA depletion