GSM9337485: B_J0_H; Homo sapiens; RNA-Seq
At each time point of the experiment (namely D0 (before adding 5-FU to medium), D1, D2, D7, D14 and D16), cells were treated for 10 min with emetin (Sigma) 25µg/ml, washed twice with PBS, and harvested by trypsination. Cytoplasmic lysates were prepared by incubation of cells for 10 min in hypotonic buffer (10 mM Tris-HCl pH 7.4, 0.5 mM MgCl2, 10 mM KCl, 1X CompleteTM EDTA free protease inhibitor (Roche) and 10 U/mL RNAseOutTM (Invitrogen)) followed by addition of 0.7 % NP-40. Swelling cells are lysed by shaking in Precellys Evolution tissue homogenizer (10s, 4500rpm). Nuclei were pelleted by centrifugation at 750 g for 5 min at 4°C, and mitochondria were pelleted by centrifugation at 12,000 g for 10 min at 4°C. The protein content of the cytosolic extract was measured by Bradford assay and samples containing 2mg protein were loaded over 15–50% sucrose gradients (poured using the Gradient Master (Serlabo Technologies)). After ultracentrifugation at 38,000rpm for 105 min at 4°C on a SW41 Beckman rotor, 18 fractions of 700µl each were collected from each gradient while 254nm absorbance profiles were generated using an ISCO UA-6 detector. RNA sampless were extracted from 150 µL of each polysomal fraction using TRIzolTM LS reagent (Invitrogen) following manufacturer's instructions and suspended in RNAse-free water. A fraction of each sample was loaded on an agarose gel, to identify their RNA content, and polysomal fractions containing mRNA with more than three ribosomes were pooled. RNA-seq libraries were prepared using the Universal Plus mRNA-seq kit (Tecan Trading AG, Switzerland) according to the manufacturer's instructions. Briefly, polyadenylated RNAs were selected using oligo-dT magnetic beads. We fragmented the polyA+ RNAs using divalent cations at elevated temperature and reverse-transcribed them using random hexamers, reverse transcriptase and actinomycin D. Deoxy-TTP was replaced by dUTP during the second strand synthesis to prevent its amplification by PCR. We repaired the double-stranded cDNAs and we adenylated them at their 3' ends followed by a ligation to Tecan adaptors including UDIs. We submitted ligated cDNAs to a strand selection prior to a PCR amplification for 15 cycles and purified the PCR products using AMPure XP Beads (Beckman Coulter Genomics, Brea, CA, USA). The size distribution of the resulting libraries was monitored using a Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA) and the libraries were quantified using the KAPA Library quantification kit (Roche, Basel, Switzerland). Libraries were denatured with NaOH, neutralized with Tris-HCl, and diluted to 300 pM. We performed the clustering and sequencing on a NovaSeq 6000 (Illumina, San Diego, CA, USA) using the single-read 100nt protocol on an S2 flow cell, to generate 50 to 84 million sequences.