title: RNAseq of G2P[4] rotavirus VU08-09-11 C-3
design: For an initial passage in primary rhesus monkey kidney (RhMK) cells (P1), 0.1 ml of a 10% (w/v) homogenate of stool suspension in triplicate for a rotavirus-positive clinical specimen that had previously been sequenced was clarified, activated with 10 g/ml trypsin (Worthington) for 1 h at 37C, then diluted in serum-free EMEM to a final trypsin concentration of < 2 g/ml. Primary RhMK cells were washed three times with serum-free EMEM and adsorbed in roller tubes with each activated specimen for 1 h at 37C with slow rotation. Following absorption, inocula were removed, monolayers were washed, and fresh serum-free EMEM containing 0.5 g/ml of trypsin was added. Cells were incubated with constant rotation at 37C for up to 7 days or until cytopathic effect (CPE) was visible. Cells then were subjected to three rounds of freezing at 80C and thawing prior to storage at 4C. In two to four subsequent passages, 0.2-1 ml of lysate from each previous lineage and passage was activated with 1 g/ml trypsin and used as the inoculum for adsorption. The presence of rotavirus in lysates was determined by ELISA. After three to five passages in primary RhMK cells, 0.5 ml of rotavirus-positive lysates were activated with 1 g/ml trypsin for 1 h at 37C. Confluent monkey kidney (MA104) cell monolayers in T25 flasks were washed with serum-free EMEM and adsorbed with 0.5 ml of P3 or P5 RhMK cell lysates for 1 h at 37C with occasional rocking. Following absorption, inocula were removed, monolayers were washed, and fresh serum-free EMEM containing 0.5 g/ml of trypsin was added. Cells were incubated at 37C for up to 7 days or until CPE was visible. Cells then were subjected to three rounds of freezing at 80C and thawing prior to storage at 4C. In nine subsequent passages, lysate from each previous passage was activated with 1 g/ml trypsin and used as the inoculum for adsorption. The presence of rotavirus in lysates was determined by ELISA, typically following P3, P6, and P10. For passages subsequent to ELISA, a 1 ml inoculum was used for lysates with values < 1; 1 ml of a 1:10 diluted inoculum was used for lysates with values between 1 and 2; and 1 ml of a 1:100 diluted inoculum was used for lysates with values > 2. Two  0.25 ml aliquots of each of MA104 P10 rotavirus-positive culture lysate were treated with 1 l of DNase I for 30 min at 37C then with EDTA to a final concentration of 5 mM to inactivate DNase I prior to RNA extraction using TRIzol LS (Invitrogen) according to manufacturer instructions. RNA pellets were resuspended in RNase-free water and incubated in a heat block set at 55C for 5-10 min, with small aliquots set aside for aside for RNA quantitation by Qubit. RNA library preparation was conducted using 5 l of input RNA and the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), according to the manufacturers instructions. Briefly, RNA was fragmented prior to first-strand and second-strand synthesis, AMPure XP Bead clean up, and end repair. PCR enrichment of adaptor ligated DNA was conducted using NEBNext Multiplex Oligos for Illumina (New England Biolabs). Illumina-ready libraries were sequenced by paired-end sequencing on a NovaSeq 6000 Sequencing System (Illumina).