SARS-CoV-2 Infection of Organoids: FACS and RNA Analysis

Organoids were infected with SARS-CoV-2 and dissociated and stained for FACS as described above in a BSL3-certified biosafety cabinet. Cells were fixed with 4% PFA for 1 hr at RT to inactivate virus as described above for decontamination out of the BSL3 facility. Cells were sorted for live, single cell, EPCAM⁺CD45⁻ cells using a BD FACSAria II SORP and RNA was extracted using the protocol described by Russell et. al^{78 (link)}. Following RNA extraction, quality was assessed via Agilent Bioanalyzer and cDNA was synthesized using Hs HostReponse LI Primers (Nanostring, XT-HHR-12), Coronavirus Panel Plus (Nanostring, CORONAPP-12) and Low RNA Input Kit (Nanostring, LOW-RNA-48) per manufacturer’s instructions. Following hybridization, transcripts were quantitated using the nCounter^® SPRINT Profiler. Samples were run by the LCA Genome Core at University of California, San Francisco.