RNAseq of Reovirus S200 virus particles
RNA libraries were prepared from RNA isolated from cesium chloride gradient-purified, benzonase-treated rsT1L virions. Contaminating DNA was degraded by treatment with RNase-free DNase I (New England Biolabs) for 10 min at 37C. RNA was isolated by using TRIzol LS Reagent (Invitrogen), according to the manufacturers protocol, and the concentration and quality of RNA was quantified using a Bioanalyzer (Agilent). Library preparation for Illumina sequencing was performed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) and a minimum of 5 ng of RNA, according to manufacturers instructions. Briefly, RNA was fragmented prior to first-strand and second-strand synthesis and RNAClean XP (Beckman Coulter) purification. PCR enrichment of adaptor-ligated DNA was performed using NEBNext Multiplex Oligos for Illumina (New England Biolabs) to produce Illumina-ready libraries. Libraries were sequenced by 150 base pair paired-end sequencing on a NovaSeq 6000 Sequencing System (Illumina). Assistance with quality control and next-generation sequencing was provided by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) research core.