Portions comparable to the latest monosomes, light, medium, and heavy polysomes were pooled separately
5, 5 mM MgCl2, 300 mM KCl, 2 mM DTT, 100 ?g/mL cycloheximide, 0.2% NP-40, and 40 U/?l RNAseOut (Invitrogen). Following a 30-min incubation at 4 °C in rotation, the lysed tissue samples were centrifugated for 15 min at 20,000?g at 4 °C. An aliquot of the lysate was used to quantify total RNA concentration using the Direct-zol RNA kit (R2051; Zymo, USA) according to the manufacturer’s instructions. From the clear supernatants of the lysates, 15 ?g of total RNA was loaded onto 10–50% linear sucrose gradients prepared in polysome buffer (20 mM Hepes pH 7.5, 5 mM MgCl2 and 300 mM KCl, 2 mM DTT), and centrifuged at 32,000 rpm (129,311?g) (SW40Ti rotor, Beckman) for 177 min at 4 °C. (tovább…)