GSM4675839: Acute - EV-2-POLY; Mus musculus; RNA-Seq
The cytoplasmic lysate was layered on top of a linear 10-40% sucrose gradient, ultracentrifuged (36,000 rpm, 2¼ hours, 4 °C) and fractionated using the Foxy Jr Fraction Collector with constant monitoring of absorbance at 260 nm by an ISCO UA-6 Absorbance Detector (Teledyne). Cytoplasmic (input) RNAs and polysomal RNAs from pooled fractions corresponding to four or more ribosomes were isolated using a TRIzol®- based method and purified using the Qiagen RNeasy® Minikit according to manufacturer's instructions, and analysed by RNAseq. cDNA libraries were generated using a ribodepletion method (TruSeq Ribo Profile Mammalian Kit, Illumina) from 1 µg of RNA sample. Fragment sizes were evaluated using the DNA 1000 kit and 2100 bioanalyzer (Agilent Technologies). Libraries were subjected to single-end sequencing (SE50bp) (Hiseq2500, Illumina) to generate ~30 million reads per sample using standard protocols.