title: GSM8673691: H9 cells, normoxia, total RNA, biol rep 2; Homo sapiens; RNA-Seq
library_construction_protocol: Cells (8 x 106 cells per condition) were seeded in 15cm plates 24h prior to the experiment. Media was changed before cells were exposed to normoxia or hypoxia (0.5% O2) for 24h or 48h. Cells were pre-treated with 100 μg/ml cycloheximide for 5 min and scraped in ice-cold, cycloheximide supplemented (100 μg/ml) PBS, using rubber scrapers. Cells were then pelleted in 15 ml conical tubes at 240 × g for 5 min at 4°C, and lysed on ice in 500 μl of hypotonic lysis buffer (5 mM Tris–HCl, pH 7.5, 2.5 mM MgCl2, 1.5 mM KCl, 100 μg/ml cycloheximide, 1 mM DTT, 0.5% Triton, 0.5% sodium deoxycholate) for 15 min. Subsequently, lysates were cleared through a 15 min, 20 817 × g centrifugation at 4°C. Cleared lysates were diluted with hypotonic lysis buffer to set their optical density (OD) at 260 nm to 10–20. Sucrose gradients (5–50%) were generated in polypropylene tubes (Beckman Coulter, 331372, 14 × 89 mm) using a gradient maker (Biocomp Gradient Master 108), according to the manufacturer's instructions. A volume of 500 μl was removed from the top of the sucrose gradient to allow for sample loading. Lysates were then layered over the top of linear sucrose gradients and centrifuged at 4°C for 2 h at 222 228 × g (36 000 rpm) using an ultracentrifuge (Beckman Coulter, Optima XPN-80, rotor SW41Ti), while 10% of the sample was kept for total cytosolic mRNA extraction. Ultracentrifuged samples were fractionated into 2 ml tubes using a density gradient fractionation system (Brandel, BR-188-177), resulting in 12 fractions of 800 μl each. Input samples, as well as sucrose gradient fractions, were mixed with 1000 μl of TRIzol LS reagent (Invitrogen) and kept at −80°C for subsequent RNA isolation. RNAseq libraries for total and polysome-associated mRNA fractions from T47D, H9, cells cultured under hypoxia (1% O2) and normoxia  (20% O2) were prepared using the Smart-seq2 protocol as previously described83. RNA quantity and quality was assessed by Qubit (Life Technologies) and Bioanalyzer (Agilent Technologies), and 10 ng of RNA was used as input material. After pre-amplification PCR 70 pg of cDNA underwent tagmentation in a total volume of 20 uL using the Nextera XT Kit (Illumina). Sequencing was then performed at the SciLifeLab National Genomics Infrastructure (NGI, Sweden) facility using the NovaSeq6000 (NovaSeq Control Software 1.6.0/RTA v3.4.4) with a 50-base paired-end setup using the 'NovaSeqStandard' workflow and S1 flow cell.