GSM4104563: CHO_6_hr_metabolic_labeling; Cricetulus griseus; RNA-Seq
Cells were collected by replacing the media by 1 mL Trizol at the following time-points: 0 min, 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr and 12 hr of chase. RNA was extracted following the Trizol extraction protocol, then re-extracted with phenol-chloroform as in. RNA was precipitated with sodium acetate and 100 % ethanol at -20°C, then spun down at 16,000 g for 20 min at 4°C, washed twice with 70 % EtOH and resuspended in water. DNAse I treatment was performed on duplicate RNA samples (2 x 6 µg) after addition of 5 ng of in vitro-labelled spike-in to each sample. The spike-in mix consisted of partial RNA sequences of B.subtilis LYSa and firefly luciferase transcripts, of which the gene sequences were cloned into pBluescript SK+ plasmid (pJC879 and pJC880, respectively) and transcribed using T7 RNA polymerase and 2 mM of 5-EU. Samples were depleted of ribosomal RNAs using Illumina Ribo-Zero Gold rRNA removal kit (MRZG12324), then purified using the RNA Clean up and concentrator Kit (R1015, the epigenetics company). The duplicates for each sample were then combined before RNA fragmentation by alkaline treatment at 95°C for 25 min (NaCO3 pH9.2 50 mM final, EDTA 1 mM final). The reaction was immediately stopped on ice by addition of 0.3 M NaOAc pH5.2, 2µL glycoblue and 500µL water. RNA was precipitated with isopropanol, spun at 16,000 g for 30 min at 4°C, washed with 70 % EtOH and resuspended in 15.75 µL of water. Biotinylation of the 5-EU labelled RNA was performed following the Click-iT nascent RNA capture kit protocol and precipitated at -80°C overnight. The fragmented and biotinylated RNA was purified on NuPAGE denaturing polyacrylamide gels (TBE-urea 15 % polyacrylamide, Thermo Fisher Scientific EC62152BOX), excising RNA between 20 and 70 nucleotides. After extraction from the gel with 500 µL of RNA gel extraction buffer (300 mM NaOAc pH5.2, 1 mM EDTA, 0.25% (w/v) SDS) on a rotator at room temperature overnight, samples were precipitated with isopropanol and resuspended in 10 µL of 10 mM Tris-HCL pH8.