Whole-genome sequencing of inflluenza A virus (H1N1): day 3 post-infection mouse lung
Total lung RNA from 30 mg tissue was extracted using the RNeasy Mini Kit (QIAGEN, Cat. No 74004), then reverse-transcribed using the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, Cat. No. 1708840) and the Uni12 primer (5-AGC AAA AGC AGG-3) with priming for 5 minutes at 25 degrees C, reverse transcription for 20 minutes at 46 degrees C, and reverse transcriptase inactivation for 1 minute at 95 degrees C (Bio-Rad thermocycler, Cat. No. 186-1096). Viral cDNA was then synthesized by PCR for 40 cycles using Taq polymerase (New England Biolabs, Cat. No. M0273) and primers containing Illumina Nextera Transposase adaptors. The primers were as follows: R1-Uni12 (5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG AGC GAA AGC AGG-5) and R2-Uni13 (5-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GAG TAG AAA CAA GG-3) (adaptor and barcode oligonucleotide sequences from Illumina, Inc., San Diego, CA, USA). Annealing and extension steps were performed for 30 seconds at 55 degrres C and 7 minutes at 72 degrees C respectively. Prior to sequencing, PCR products were cleaned using the QIAquick PCR Purification Kit (QIAGEN, Cat. No. 28104), and up to 20 pM DNA samples were used for library preparation. Libraries were sequenced by CGEB-IMR (Centre for Genomics and Evolutionary Biology Integrated Microbiome Resource, Halifax, NS, Canada) (http://cgeb-imr.ca)) in a 300 + 300 bp paired-end MiSeq run (Illumina 600-cycle v3 kit, Cat. No. MS-102-3003).