RNA sequencing and pathway enrichment data analysis
RNA from the whole blood of 16 MHV-1- and 16 mock-infected C3H/HeJ mice
were submitted to BGI (Cambridge, MA, USA) to perform library prep and
sequencing using their DNBseq platform. RNA quality was assessed using
Nanodrop 260/280 and 260/230 ratios and raw reads were generated as
Fastq files. We performed quality control on the raw reads using
FastQC26 and trimmed them using
BBDuk27. The mouse reference genome (GRCm39) and
corresponding annotations were downloaded from National Center for
Biotechnology Information (NCBI, Bethesda, MD, USA). The clean reads
were mapped to the reference genome using the Spliced Transcripts
Alignment to a Reference (STAR) with default
parameters28. HT-Seq (htseq-count) within the STAR
package was used to estimate gene counts29.
Differential gene expression analysis was conducted using
DESeq230, and Gene Set Enrichment Analysis (GSEA) was
performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG)
database. We employed the integrated differential expression and pathway
analysis tool (iDEP 0.96, South Dakota University, SD, USA)31 for these analyses, setting the default parameters
to a p-adjusted value of <0.1 and fold change of
>2 for identifying differentially expressed genes. The
expression patterns of identified biomarker genes were plotted with gene
plot feature in iDEP 2.0 using raw counts and standard deviation.