Ncounter flex gene expression

Relative mRNA abundance was determined by multiplexed hybridization of fluorescently labeled and barcoded gene specific probes for digital readout using the analysis system NanoString nCounter FLEX gene expression (Nanostring Technologies, Seattle, Washington, USA) with DX enablement following the manufacturer's instructions. The nCounter Human Host Response Panel was employed covering 785 genes across more than 50 pathways to evaluate the innate and adaptive immune responses to SARS-CoV-2 infection and treatment with the p38 inhibitors. 5 × 10⁵ Calu-3 cells were solvent-treated but non-infected (non-infected control), infected with SARS-CoV-2 at MOI 0.001 in the presence of the solvent (control), or infected and inhibitor-treated (PH and VX) for 48 h. 350 ng total RNA was hybridized to the reporter and capture probes according to the CodeSet Hybridization Setup for 18 h at 65 °C. Hybridized samples were loaded into the nCounter prep station for post-hybridization processing and analyzed with the nCounter Digital analyzer. The NanoString nCounter nSolver software (version 4.0), with an additional NanoString Advanced Analysis Module (version 2.0.134) was used to perform quality control assessment, normalization and cartridge correction. Differential gene expression analysis was performed as described in statistical analysis. Expression data are shown in Supplementary Table 2.