NRVMs were collected at 48 h after infection with adenoviruses for lacZ, Nrf1, and Nrf2. RNA was extracted using the RNeasy Mini Kit (QIAGEN, 74104) according to the manufacturer’s protocol. An Agilent 2100 TapeStation was used for RNA quality analysis. Stranded mRNA-seq libraries were generated using the KAPA mRNA HyperPrep Kit (Roche, KK8581) following the manufacturer’s protocol. Sequencing was performed on an Illumina NextSeq 500 system using a 75-bp, high-output sequencing kit for single-end sequencing.
For data analysis, quality control of RNA-seq data was performed using the FastQC Tool (Version 0.11.4). Sequencing reads were aligned to rat Rnor 6.0 reference genome using HiSAT2 (Version 2.0.4) with default settings and -rna-strandness F55 (link). After removal of duplicate reads using Samtools v0.1.1856 (link), aligned reads were counted using featurecount (Version 1.6.0) per gene ID57 (link). Differential gene expression analysis was performed with the R package edgeR (Version 3.20.5) using the GLM approach58 (link). For each comparison, genes with more than 1 count per million (CPM) in at least three samples were considered as expressed and were used for calculating the normalization factor. Cutoff values of absolute fold-change >1.5 and false discovery rate <0.05 were used to select DEGs between sample group comparisons.
For data analysis, quality control of RNA-seq data was performed using the FastQC Tool (Version 0.11.4). Sequencing reads were aligned to rat Rnor 6.0 reference genome using HiSAT2 (Version 2.0.4) with default settings and -rna-strandness F55 (link). After removal of duplicate reads using Samtools v0.1.1856 (link), aligned reads were counted using featurecount (Version 1.6.0) per gene ID57 (link). Differential gene expression analysis was performed with the R package edgeR (Version 3.20.5) using the GLM approach58 (link). For each comparison, genes with more than 1 count per million (CPM) in at least three samples were considered as expressed and were used for calculating the normalization factor. Cutoff values of absolute fold-change >1.5 and false discovery rate <0.05 were used to select DEGs between sample group comparisons.
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