Pgfip piston gradient fractionator

Cell pellets were lysed in 500 ul of lysis buffer (20mM Tris-Cl, pH 8.0, 150mM NaCl, 15mM MgCl2, 1% Triton-X 100, 40U Turbo DNase I, 40mM NEM, 1mM DTT, EDTA-free protease inhibitor cocktail in DEPC treated water) followed by vigorous pipetting and incubated on ice for 15min. The cell lysates were centrifuged at 15,000 rpm for 10min at 4°C and the supernatant was transferred to a new microcentrifuge tube. Total RNA concentration of each lysate was determined using a nanodrop (Thermo Scientific). 500ug of total RNA was digested with 3.5ug/ml of RNaseA for 15min at 25°C on a thermomixer (Eppendorf) at 500rpm. The digestion was stopped with 166.5U of SUPERaseIn RNase Inhibitor. Samples were fractionated over a 10–30% sucrose gradient containing 150ug/ml cycloheximide (prepared on Gradient Master 108 (Biocomp): 1min 54s, 81.5 degrees, 16rpm). Samples were centrifuged at 41,000rpm for 2 hr at 4°C in an SW41i rotor. 1ml fractions were collected using a PGFip piston gradient fractionator (Biocomp). Protein fractions were precipitated overnight with 10% TCA at 4°C, followed by three ice-cold acetone washes. Pellets were dried in Vacufuge plus (Eppendorf) at room temperature for 5 min. Pellets were then resuspended in Laemmli sample buffer with β-mercaptoethanol, heated at 95°C for 10 min.