GSM7051328: r08_40wpi; Rattus norvegicus; OTHER
Viral RNA was extracted using TRIzol, followed by standard on-column purification procedure. The wole ORF was reverse transcribed to generate viral cDNA. Samples were pre-incubated at 65ºC for 5 min prior to the reaction. RT was carried out at 50ºC for 2 h in the presence of RNase inhibitors using 0.1 μM primer TS-O-00319 (GCTTCCTGGAGCGGGCTAGATACTG). Amplification of the complete ORF was performed using Q5 High-Fidelity Hot start DNA Polymerase (NEB), including high GC Enhancer, and the primer pair TS-O-00318 (CCAAGCCCCAATGCCGTCCGGCACCGCTGCCCTTTTCGG) and TS-O-00361 (AGGTGAAGGGGGCATCGATG). Amplified DNA was purified using DNA Clean & Concentrator (Zymo Research). Libraries were constructed using the NebNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs), using dual indices. cDNA input: 25ng, adapter ligation, bead-based size selection (AMPure XP beads; 200-350 bp), PCR enrichment using i7/i5 index primer set. Enriched libraries were washed twice using AMPure XP beads for complete depletion of residual adapter. Libraries were sequenced paried-end on a Illumina Miseq using 500 PE V2 kit (Illumina).