GSM4189767: mESCs, totRNA_DMSO_1; Mus musculus; RNA-Seq
4SU-seq was performed essentially as described previously (Rabani et al, 2011 PMID: 21516085). Briefly, 4-thiouridine (4SU; Sigma - catalogue no. T4509) was added to ESCs in culture to a final concentration of 500 µM and incubated at 37°C for 20 min. Cells were harvested by trypsinisation and washed twice with PBS at RT. Total RNA was isolated from 7x10e6 cells using trizol according to the manufacturer's instructions (Invitrogen - catalogue no. 15596026). Following precipitation, purified RNA was resuspended in 100 μl RNase-free water and DNase treated using the TURBO DNA-free kit according to the manufacturer's instructions (Invitrogen - catalogue no. AM1907M). Residual inactivation beads were removed by spinning the RNA sample through a QIAshredder column at 1000 g for 1 min (Qiagen- catalogue no. 79654). 2μg of RNA input was retained for direct sequencing of the total RNA pool and 30 μg was incubated for 1.5 h at RT with 100 μg of Biotin-HPDP (Pierce - catalogue no. 21341; reconstituted in Dimethylformamide at 1 mg/ml) in 1x Biotinylation Buffer (10 mM Tris pH 7.4 and 1 mM EDTA) to a total volume of 500 μl. Uncoupled biotin was removed through two consecutive rounds of 1:1 v/v chloroform extraction followed by isopropanol/NaCl precipitation. RNA was resuspended in 100 μl of RNase free water and mixed 1:1 w/w with µMacs Streptavidin beads (miltenyi - catalogue no. 130-074-101) and incubated for 15 min at RT with rotation. The RNA / bead mixture was applied to a µMacs column following pre-equilibration with wash buffer (100 mM Tris pH 7.5, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). The captured beads were then washed with 3 x 900 µl of 65 oC wash buffer and 3 x 900 µl RT wash buffer. RNA was then eluted from the column by adding two consecutive rounds of 100 mM DTT. The eluate was added to 700 µl Buffer RLT (RNeasy MinElute Cleanup Kit; Qiagen catalogue no. 74204) and then purified according to the manufacturer's instructions. Prior to library preparation, ribosomal RNA was depleted from both the total and purified nascent RNA using the low Input RiboMinus Eukaryote System v2 kit according to the manufacturer's instructions (Ambion - catalogue no. A15027). Libraries were constructed using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina according to the protocol for ribosome depleted RNA and with a 11 min RNA fragmentation step (NEB - catalogue no. E7760). Library PCRs were supplemented with 2x SYBR dye (Sigma – catalogue no. S9430) so that amplification could be monitored by quantitative PCR on a Roche lightcycler 480. To allow for sample multiplexing, PCRs were performed using index primers (NEBNext Multiplex Oligos for Illumina - Set 1. Catalogue no. E7335) and amplified to linear phase. Size selection purifications following the ligation and amplification PCR steps were performed with 1x and 0.9x reaction volumes of Agencourt AMPure XP beads (Beckman Coulter - A63880). Purified libraries were combined as an 8 sample equimolar pool containing the indexes 5-12 and sequenced on an Illumina NextSeq on a single high-output flow cell (paired-end 75 bp reads).
GEO Accession
GSM4189767