GSM2367470: MCF-7 4SU mock replicate 2 IR 1h; Homo sapiens; RNA-Seq
Total RNA was extracted using miRNA easy kit (Qiagen) using manufacturer's instruction. Total RNA (60 µg) was treated with 12 U Turbo DNase (Thermo Fisher) in 350 µl reaction volumes for 30 min at 37ºC. To remove the enzyme, RNA was phenol-chloroform extracted, followed by isopropanol precipitation and resuspension in water. We next performed 4SU biotinylation as previously described (Gregersen et al., 2014a; Radle et al., 2013) with minor modifications. Briefly, 4SU biotinylation reactions were carried out at room temperature for 3 h in 250 µl reaction volumes containing 10 mM Tris pH 7.5, 5 mM EDTA, 50 µg MCF-7 total RNA, 250ng S2 cell RNA (200:1 ratio) and 100 µg EZ-Link biotin-HPDP (Thermo Fisher). Afterwards, RNA was phenol/chloroform extracted in phase-lock tubes, isopropanol precipitated and resuspended in 100 µl of water. RNA was next denatured at 65ºC for 10 min, cooled on ice and incubated for 15 min at room temperature with 150 µl streptavidin-conjugated magnetic beads (µMACS, Miltenyi Biotec). To isolate 4SU containing RNA, the mixtures were applied to µColumns, washed 3 times in washing buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween20) and eluted twice in 100µl of 0.1M DTT. Cleanup was performed by RNAeasy MinElute kit (Qiagen) with elution in 14 µl of water. Typical yields were in the range of 25 - 50 ng. Unstranded Truseq mRNA kit v2 was used according to the manufacturer's instruction to prepare cDNA libraries using 10 µl of starting 4SU-enriched RNA. Clustering and amplification of cDNA libraries was performed according to manufacturer's instruction. Massively parallel sequencing was carried out on HiSeq2000 Illumina instrument using 1x101+7 cycles. Illumina Truseq RNA v2 kit (unstranded)