GSM5138256: PolysomalMNV-10hrs-5 [P_MNV_10_5]; Mus musculus; RNA-Seq
RAW264.7 cells infected with UVi(MNV) or MNV at MOI 10 for 6 and 10h were treated with 100µg/mL of cycloheximide for 3min at 37°C to lock the translating ribosomes onto the transcripts before being placed on ice, washed twice with cold PBS(-) containing 100µg/ml CHX and lysed on plate with 1mL of cold lysis buffer (15mM Tris-HCl, pH 7.5, 300mM NaCl, 15mM MgCl2, 100µg/mL CHX, 1% (v/v) Triton X-100, 200u RNasin for 5min. The post-nuclei supernatants were aliquoted in 2 tubes for Total and Polysomal RNA isolation. 500µl of each lysate were loaded onto 10mL 10-60% discontinuous sucrose gradients (15mM Tris, pH 7.5, 300 mM NaCl, 15mM MgCl2, 100µg/mL CHX, 1mg/mL Heparin) and spun at 38krpm for 2h at 4°C using the SW41Ti rotor. Gradients were fractionated using the fraction collector FoxyR1 in 10 fractions of 1mL. Total lysates and gradient fractions were denatured in 1.5mL of 7M GnHCl, ethanol-precipitated and resuspended in 30µL of RNAse free water. The fractions containing the polyribosomes were identified by their composition in 40 and 60S ribosomal RNA on 2% agarose gel, pulled together, further purified by two rounds of LiCl precipitation and resuspended into 30µL of RNAse free water. Obtained RNA were quality controlled using the Agilent Bioanalyser and quantified on Nanodrop. 1µg of RNA per sample were sent to the Oxford Genomics Centre (Welcome Trust Centre for Human Genetics, Oxford, UK) which generated the cDNA libraries by enrichment in polyA-tailed messenger RNA and performed the RNA sequencing on Illumina HiSeq4000 machine, 75bp PairEnds protocol with multiplexing of the samples.