Quantstudio 7 system

Mid-turbinate nasal swabs collected from individuals participating in the study were analyzed using RT-PCR for the detection of nucleic acid from SARS-CoV-2. The RT-PCR assay used for this study was based on the initial assay implemented for the UNC Respiratory Diagnostic Clinic by the Clinical Microbiology and Molecular Microbiology Laboratories at UNC Hospitals. In brief, RNA was extracted using the Roche Diagnostics MagNA Pure MPC large volume isolation system (Roche Diagnostics, Indianapolis, IN, USA) and NucleoSpin Isolation kit (MACHEREY-NAGEL GmbH & Co. KG, Germany). Samples were then quantified, reverse transcribed (SuperScript 3, ThermoFisher, USA) on a thermocycler. Using primers based on the Respiratory Diagnostic Clinic assay and human RNA control primers, amplicons were amplified and then quantified using a ThermoFisher QuantStudio 7 system. While based on the clinical assay, this RT-PCR assay did not have Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA); therefore, all positive results were referred for confirmatory testing using an EUA-approved PCR test in a Clinical Laboratory Improvement Amendments (CLIA) approved laboratory 3–10 days after the research sample was collected.

Frozen lung tissue was ground to a fine powder using a mortar and pestle. RNA from isolated alveolar macrophages or lung tissue were purified using the RNeasy mini kit as per manufacturers instruction (Qiagen, GMH Germany). The NanoDrop200 (Thermo Fischer Scientific, United States) was used to determine the concentration and purity of extracted RNA and the High-Capacity RNA to cDNA kit (Thermo Fischer Scientific, United States) was used to generate complementary DNA (cDNA). RT-qPCR was performed using the QuantStudio 7 system (Thermo Fischer Scientific, United States), TaqMan primers and the Taqman Fast Advanced Master Mix, as per manufacturer’s instructions. The relative expression of each gene was normalised against the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) and determined using the ΔΔCt value method, as previously described (Wang et al., 2018 (link); Wang et al., 2019 (link)).