alias: GSM4544780
title: GSM4544780: WT_10min_tot; Mus musculus; RNA-Seq
library_construction_protocol: 4sU labelling and extraction protocol: Five million DTCM23/49 XY mESCs (WT and miRNA depleted) were seeded and allowed to grow to 70-80% confluency (approximately 1 day). 4sU was added to the growth medium (final concentration of 200 µM) and the cells were incubated at 37 ºC for 10 minutes and extracted using Trizol, according to manufacturer instructions. RNA was DNase treated using RNeasy on column digestion according to manufacturer's instructions. 100 µg of RNA was incubated for 2 h at room temperature with rotation in 1/10 volume of 10X biotinylation buffer (Tris-HCl pH 7.4, 10 mM EDTA) and 2/10 volume of biotin-HPDP (1mg/ml in Dimethylformamide (Thermo Fisher, 21341)). Following biotinylation, total RNA was purified through phenol:chloroform:isoamyl alcohol extraction and precipitated with equal volume of Isopropanol and 1/10 volume of 5M NaCl. Precipitated RNA was washed once with 75% Ethanol and resuspended in in DEPC-treated H2O. Equal volume of biotinylated RNA and pre-washed DynabeadsTM MyOneTM Streptavidin T1 beads were mixed and incubated at room temperature for 15 minutes under rotation. The beads were then separated using a DynaMagTM-2 Magnetic stand. The supernatant (that contains unlabeled preexisting RNA) was placed at 4oC until precipitation. Beads were washed and biotinylated RNA dissociated from streptavidin coated beads by treatment with 100 mM 1,4-Dithiothreitol for 1 minute, followed by 5 minutes in RTL buffer. Beads were separated from the solution using DynaMagTM-2 Magnetic stand and the RNA was recovered from the supernatant was extracted using Qiagen RNeasy Mini Kit according to the manufacturer's instructions. Preexisting RNA was precipitated with equal volume of Isopropanol and 45 minutes centrifugations at 15 000 g (4oC). Preexisting RNA pellet was washed with 75% Ethanol and resuspended in DEPC-treated H2O. Metabolic labelling was carried out twice (2 biological replicates). Total RNA libraries were prepared from 10 ng of preexisting RNA and newly transcribed RNA using Ovation® RNA-Seq and sequenced on Illumina HiSeq 2500.
identifiers.namespace: GEO
identifiers.id: GSM4544780