Bulk RNA-seq of young and aged livers following influenza A virus infection

Aging is known to alter the host repsonse to influenza infection. Here, we use bulk RNA sequencing (bulk RNA-seq) to identify cellular changes in the livers of young (16-week-old) and aged (80-week-old) mice following influenza infection. Mice were infected with influenza A/PR8/34 (~50 PFU/mouse) to establish acute infection. Infections were performed by intranasal (i.n.) administration under anesthesia as described before (Sun et al., 2009). Mice were then euthanized and the right ventricle was perfused with 10 mL cold DPBS (Corning). For bulk RNA-seq, pieces of liver were harvested after ventricular perfusion and snap frozen on dry ice. RNA was extracted using an RNAEasy Mini Kit (Qiagen). Reverse transcription was performed and the resultant cDNA was enzymatically fragmented and indexed using a Nextera XT DNA Library Preparation Kit (Illumina). Barcoded cDNA libraries were sequenced on an Illumina NextSeq 500 instrument using a NextSeq 500/550 High Output Kit v2 (75 cycles) (20024906, Illumina) with the following cycle counts: 37 (read 1), 8 (index 1), 8 (index 2), 37 (read 2). Demultiplexing was performed using bcl2fastq (v2.20, Illumina). Alignment was performed using Salmon (v1.4.0) (Patro et al., 2017) and the GRCm39 reference genome (Ensembl) with default settings. Downstream analysis was performed in R using DESeq2 (v1.28.1) (Love et al., 2014).