GSM6067260: DF2.ADAR_Polysome_Rep1; Homo sapiens; RNA-Seq
Cell lysate (10mM HEPES pH.7.4, 150mM KCl, 1mM MgCl2, 2% NP-40, 1mM DTT, 100 µg/mL cycloheximide, 40U/mL RNaseOUT, cOmplete Mini EDTA-free protease inhibitor (Roche)) was separated on a 10-50% linear sucrose gradient by centrifugation at 28.000g for 3h at 4C in a SW28Ti rotor (Beckman). Fractions containing polysomes (>2 ribosomes) were pooled and total RNA was precipitated with ethanol. For each sample 150ng of total RNA was used for library preparation. Library construction was performed using the NEBNEXT Ultra II Directional RNA-Seq kit and the NEBNext rRNA Depletion Kit.