GSM6523402: Ribo-seq MCMV 2hpi - Cycloheximide - Rep 2; Mus musculus; OTHER
Cells were snap frozen followed by cell lysis in presence of cycloheximide. Following removal of cell nuclei by centrifugation and stringent RNAse digest, ribosomes were pelleted through a sucrose cushion. Translated RNA fragments were recovered by size selection. Translated RNA fragments were cloned following the protocol described by Stern-Ginossar N et al. Science 2012. rRNA depletion involved additional biotinylated oligos to improve depletion. To efficiently remove all biotinylated oligos complementary to specific rRNA sequences, two rounds of streptavidin depletion were performed. cDNA libraries were amplified by PCR and sequenced (HiSeq 50nt reads).