Polysome Fractionation and RNA Analysis

Total polysome was isolated and fractionated using sucrose gradient fractionation as described elsewhere [43 (link)]. Briefly, cells were incubated with 100 μg/ml of cycloheximide (Sigma) at 37 °C for 10 min. The cells were lysed in polysome extraction buffer (10 mM HEPES pH 8.0, 5 mM MgCl₂, 25 mM KCl, 1% Triton X100, 1 mM DTT, 1% sodium deoxycholate, 5 mM vanadyl ribonucleoside complex, 1X protease inhibitor cocktail (Roche) containing 100 μg/ml cycloheximide). The cytoplasmic extract was isolated, and total RNA was quantitated using NanoDrop. An equal amount of cytoplasmic lysate was fractionated on a sucrose gradient (10–50%) and ultracentrifuged at 190,000 g for 90 min at 4 °C in Sorvall WXUltra100 (Thermo Fischer Scientific) with AH650 rotor. Total RNA was isolated and analyzed from each volume 500 μl fraction and percent of RNA was calculated.