Input RNA for hypoxia (Total)
Cells were pre-treated with cycloheximide (100 µg/ml, Sigma-Aldrich, St. Louis, MO) for 5 min before lysis and collected in cold PBS containing 100 µg/ml cycloheximide. Cells were lysed in RSB-150 (10mM Tris-HClpH 7.4, 3 mM MgCl2, and 150 mM NaCl) containing100 µg/ml cycloheximide, 40 µg/ml digitonin (Calbiochem), 20 U/ml RNasin (Promega), and 1X protease inhibitor cocktail (Thermo Fisher Scientific). After incubation on ice for 5 min, cells were disrupted by passage through a 26-gauge needle five times. Cytoplasmic extracts were collected by centrifugation at 3,000 × g for 2 min, and clarified by further centrifugation at 11,000 × g for 15 min. The samples were loaded on a linear gradient of 15-40% sucrose and centrifuged at 38,000 rpm for 3 h in a Beckman SW41 rotor. After centrifugation, 11 fractions were collected from each gradient. Total RNA was isolated using phenol-chloroform extraction followed by ethanol precipitation.