Illumina HiSeq 1500 sequencing; Large-scale study of total and polysomial mRNA after DDX6 depletion in HEK293 cells
Large-scale study of total and polysomial mRNA after DDX6 depletion in HEK293 cells
Human embryonic kidney HEK293 cells  were maintained in DMEM supplemented with 10% (v/v) fetal calf serum. siRNA transfections were performed using Lipofectamin 2000 (Life Technologies, France) according to the manufacturer's protocol. Cells were harvested 48h post-transfection HEK 293 cells were harvested in ice-cold PBS, lysed for 10 minutes in ice-cold polysome lysis buffer (20 mM HEPES pH 7.4, 250 mM KCl, 10 mM MgCl2, 5 mM DTT, 0.5 % (v/v) NP40) supplemented with EDTA-free protease inhibitor cocktail (Roche Diagnostics) and 20 units/mL RNaseOut (Promega). Cell lysates were centrifuged at 500 g for 5 min to eliminate nuclei. Supernatant corresponding to 106 cells was reserved for total RNA extraction, while the remaining sample (~8.106 cells) was separated through a 36 mL 10-50 % (w/v) sucrose gradient in polysome buffer (20 mM HEPES pH 7.4, 250 mM KCl, 20 mM MgCl2, 2mM DTT). After centrifugation at 26 000 rpm for 4 h at 4C in a SW-27 rotor (Beckman Coulter), 28 fractions were collected by upward displacement with the ISCO gradient fractionation system (Teledyne Osco) connected to a UV detector to continuously monitor the absorbance at 254 nm. Total and polysomial RNA was extracted using the SV Total RNA Isolation System (Promega) according to the manufacturers instructions Library preparation was performed at the Ecole Normale Superieure Genomic Platform (Paris, France). Ribosomal RNA was depleted from 1 g of total or polysomial RNA using the Ribo-Zero kit Human/Mouse/Rat (Epicentre). Libraries were prepared using the strand specific RNASeq library preparation ScriptSeq V2 kit (Epicentre), and multiplexed by three on flow cell lanes.