Total rna prep with ribo zero plus kit

Right lung caudal lobe was harvested from the animals and homogenized in trizol. RNA purification was performed with the kit RiboPure RNA Purification Kit following manufacturer’s instructions. Sequencing libraries were generated with the Illumina Total RNA Prep with Ribo-Zero Plus kit. These libraries were sequenced on an Illumina NovaSeq 6000 using an S4 flow cell, generating paired-end 100 bp reads. Adapter sequences were trimmed from reads with Cutadapt [85 ], then pseudoaligned by Kallisto [87 (link)] to the Golden Hamster transcriptome. The transcriptome index was built from the MesAur1.0 genome assembly and gene annotation from Ensembl release 100. Differentially expressed genes were identified using DESeq2 [86 (link)]. Cell types were deconvoluted from bulk RNA-seq profiles with CIBERSORTx [84 (link)]. A signature matrix was built from a single-cell Golden Hamster lung dataset [40 (link)], which was randomly down-sampled to 100 cells of each type. Cell fractions were imputed using default parameters and a normalized count matrix as input. Analysis were performed and graphs were performed in R, using the packages “DESeq2” (v 1.38.3), “pheatmap” (v 1.0.12) and “patchwork” (v 1.1.2). Viral RNA reads were retrieved from RNA-seq data aligning them in with variant-specific references for each group.