The transcriptional and translational landscape of equine torovirus

Hazel Stewart; Katherine Brown; Adam M. Dinan; Nerea Irigoyen; Eric J. Snijder; Andrew E. Firth

Cells were either not pretreated or, where stated, were treated with a final 555 concentration of 100 μg/mL cycloheximide (CHX) for 2 minutes (Sigma-Aldrich) or 2 556 μg/mL of harringtonine for 3 minutes (LKT Laboratories) followed by CHX for 2 557 minutes, before flash-freezing. RNA and ribosomes were harvested according to 558 previously published protocols (15, 51) with minor modifications. Following either 559 RPF or RNA isolation, duplex-specific nuclease was not utilised but instead rRNA was 560 depleted with the RiboZero [human/mouse/rat] kit (Illumina). Libraries were 561 prepared and sequenced using the NextSeq500 platform (Illumina). were then mapped sequentially to the rRNA, vRNA, mRNA and ncRNA databases 580 using bowtie version 1.2.1.1 (56), with parameters -v 2 --best (i.e. maximum 2 581 mismatches, report best match), with only unmapped reads passed to each following 582 . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/296996 doi: bioRxiv preprint stage. Reads that did not align to any of the aforementioned databases were then 583 mapped to the host gDNA using STAR version 2.5.4a (57), again allowing a maximum 584 of 2 mismatches per alignment. Remaining reads were classified as unmapped. 585