Mitotic RiboSeq of HeLa cells: DENR_KO 2.11
For RiboSeq, cells were briefly rinsed with ice-cold PBS containing 10mM MgCl2, 400uM cycloheximide, then spun down and lysed with 150ul of lysis buffer (250mM HEPES pH 7.5, 50mM MgCl2, 1M KCl, 5% NP40, 1000uM CHX) per sample. After brief vortexing, lysate was clarified by centrifuging for 10 minutes at 20.000g at 4C. Approximate RNA concentration was measured using Nanodrop szstem and 1ul of Ambion RNAse 1 was added per 120ug of measured RNA. Lysates were incubated with RNAse for 5 minutes on ice. Lysates were then pipetted onto 17.5-50% sucrose gradients and centrifuged at 35.000 rpm for 3.5 hours in Beckman SW40 rotor. Gradients were fractionated using a Biocomp Gradient Profiler system and 80S fractions were collected for footprint isolation. RNA was isolated from these fractions using Acid-Phenol extraction and analysed on an Agilent Bioanalyzer system to assess RNA integrity.
AS-590215-LR-56173