Profiling of Ribosome-Associated Transcripts in EToV-Infected Cells

ED cells were infected with EToV for 1 h in serum-free medium (MOI of 0.1) and flash-frozen in liquid nitrogen at 8 h.p.i. prior to either RNA isolation or ribosome purification for profiling. Cells were either not pretreated or, where stated, were treated with a final concentration of 100 μg/ml cycloheximide (CHX) for 2 min (Sigma-Aldrich) or 2 μg/ml of harringtonine for 3 min (LKT Laboratories), followed by CHX for 2 min before flash-freezing. RNA and ribosomes were harvested according to previously published protocols (17 (link), 58 (link)), with minor modifications. Following either RPF or RNA isolation, duplex-specific nuclease was not utilized but instead rRNA was depleted with the RiboZero [human/mouse/rat] kit (Illumina). Libraries were prepared and sequenced using the NextSeq500 platform (Illumina).