RNA-seq

Total RNA from mock-treated or vaccinia-infected cell lines was extracted using Trizol reagent (Invitrogen). Illumina Stranded TruSeq total RNA kit was used for library preparation. Total RNA depleted of ribosomal RNA using Ribo-Zero was converted to cDNA, and the resulting single-read 75 bp library was sequenced on an Illumina Hi-Seq instrument. The demultiplexed and de-indexed FASTQ files were processed using the Galaxy platform (https://usegalaxy.org/; Afgan et al., 2018). Briefly, FASTQ files were quality-control checked using FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and aligned to the human genome (Hg38) using HISAT2 (Kim et al., 2015). The aligned BAM files were used for counting gene features using FEATURECOUNTS (Liao et al., 2014). The DEBROWSER package (Kucukural et al., 2019) was used for differential gene expression analysis using DESEQ2 (Love et al., 2014) and visualization of volcano plots and heatmaps. FUNRICH (Pathan et al., 2015) was used for generating gene set Venn diagrams and to identify overlapping gene sets. To map reads to the vaccinia genome, the FASTQ files were aligned with custom indexed vaccina WR genome (AY243312.1) using BWA (Li and Durbin, 2009). HTSeq counts (Anders et al., 2015) was used to identify mapped reads with genomic features. DEBROWSER was used as described above for identifying differentially expressed vaccinia genes. Heatmaps and scatterplots were generated using Graphpad Prism (9.0.0). Gene ontology analysis was performed using Funrich (Fonseka et al., 2020).