Sybr green fluorescence

Total RNA was extracted with TRIzol reagent (Invitrogen, USA), and the first-strand cDNA was generated with a reverse transcription (RT) kit (Promega, USA). Quantitative real-time PCR was performed to measure gene expression using different pairs of primers (Table S1). Quantitative real-time PCR was conducted using an Applied Biosystems with SYBR Green fluorescence (Promega, USA) for 40 cycles, which consisted of heat denaturation at 95°C for 30 s and annealing extension at 60°C for 60 s. All relative gene expression levels were normalized to GAPDH.

Analysis of ISGs was performed as previously described (Luksch et al., 2019 (link)). Total RNA from mouse lungs was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity were determined spectrophotometrically. 2 µg RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase, Rnasin, and dNTPs (all Promega) in 35 μl reaction according to the manufacturer’s instructions. qRT-PCR was performed using Quantstudio 5 (Thermo Fisher Scientific) and GoTagqPCR Master Mix with SYBR green fluorescence (Promega). PCR primer sequences were retrieved from online Primer Bank database (Spandidos et al., 2010 (link)). Expression of genes was normalized with respect to each housekeeping gene using the ^ΔΔCt method for comparing relative expression (upregulation > 2.0 and downregulation < 0.5).