title: SARS-CoV-2 whole genome sequencing after mus musculus challenge: intracranial vs intranasal challenge
design: K18-ACE2 mice were challenged intranasally with 6x10^3 plaque forming units (PFU) of SARS-CoV-2/human/USA/USA-WA1/2020 and euthanized at 5-days post infection. Whole lung and brain tissue was dissected and RNA extracted for viral whole genome sequencing. cDNA synthesis was performed with SuperScript IV (Thermo 18090200) using random hexamers according to manufacturers specifications. Direct amplification of viral genome cDNA was performed using the Artic Network version 4 primers. Sequencing library preparation of amplicon pools was performed using the SeqWell plexWell 384 kit (Seqwell PW384A) per manufacturers instructions. Pooled libraries were sequenced on the Illumina MiSeq using the V2 500 cycle kit. To generate consensus sequences, reads were trimmed to remove adapters and low-quality sequences using Trimmomatic v0.36. Trimmed reads were aligned to the reference genome sequence of SARS-CoV-2 (accession MN908947.3) using bwa v0.7.15.