A Mouse Model of SARS-CoV-2 induced Acute Lung Injury (ALI) in Standard Laboratory Mice
RNA isolates were purified using a Direct-zol RNA Kit (Zymo Research) following manufacturers suggested protocol and quantified by Nanodrop (ThermoFisher Scientific).  dsDNA was synthesized by random priming with Random Primer 9 (New England BioLabs) on 500-1000 ng of each isolates RNA and reverse transcribed using Super Script II (Signma-Aldrich) to make cDNA followed by second strand synthesis using NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England BioLabs) following the manufacturers suggested protocols.  dsDNA was quantified using Qubit dsDNA HS Assay Kit (ThermoFisher Scientific).  Libraries were prepared using Nextera XT DNA Library Preparation Kits (illumina) and sequenced on a NovaSeq 6000 System (illumina) with paired end reads (2 x 151).  Passage 1 reads were de novo assembled using CLC Genomics Workbench v12 (Qiagen) to confirm initial viral sequence.  Single nucleotide polymorphisms (SNPs) were determined by aligning trimmed reads to the initial viral sequence using Bowtie2 v2.3.4.1 and Samtools v1.10 and the aligned reads were visualized using Integrative Genomics Viewer v2.7.2.  SNPs were called based on mutations noted versus background error rate.  All locations in question had over 5000x coverage.