GSM2791567: 40HipPPPoly; Homo sapiens; RNA-Seq
Polysomal mRNA was obtained by 10%–50% sucrose gradient sedimentation. Upon hypoxia or normoxia, with and without PP242 treatments, cells were washed twice with cold 1× phosphate-buffered saline (PBS) and lysed by incubation for 10 minutes on ice in polysome buffer: 1.5 mM KCl, 5 mM Tris-HCl pH 7.4, 2.5 mM MgCl2, 1% Triton X-100, 1% Na-deoxycholate, 100 μg/ml cycloheximide, 2.5 μl/mL RNAaseOut and 1× Complete Roche Protease Inhibitor. The cell lysate was centrifuged at 12,000 × g for 15 minutes at 4°C. One microgram of total protein from the supernatant was loaded onto a 10%–50% sucrose gradient, made with the BioComp Gradient Maker, and ultracentrifuged at 37,000 rpm (SW40 rotor) for 150 minutes at 4°C. The sucrose gradient was fractionated with the ISCP UV gradient fractionation system (BioComp), connected to a UV detector to monitor absorbance at 254 nm, and the polysome profile was recorded. Twelve fractions of 900 μl each were isolated and RNA was extracted using phenol:chloroform and ethanol precipitation followed by an RNeasy Mini Kit (Qiagen) for DNase treatment according to the manufacturer's instructions. Both total RNA and polysome-bound mRNA were analyzed on an Agilent Bioanalyzer to assess RNA integrity. The RNASeq libraries were prepared from total RNA using the TruSeq™ RNA Sample Prep Kit v2 (Illumina Inc.,).