GSM7817964: A549-ACE2_infected_total_5; Homo sapiens; RNA-Seq
At 24 h post infection, cells were detached with trypsin, fixed in 4% PFA for 30 min on ice and stained for spike protein as described above for flow cytometry, with RNasin added to FACS buffer (1:100 dilution) just before use to prevent RNA degradation. Infected cell samples were resuspended in PBS 2%, 25 mM Hepes, 5 mM EDTA (sorting buffer) and sorted at 4°C on a FACSAria Fusion4L Sorter into infected (presence of S protein expression) and bystander (absence of viral protein expression) cell populations. Cells were collected in FBS-coated tubes containing buffer with RNasin to minimize RNA degradation. After sorting, cells were pelleted at 500g for 5 min at 4°C and RNA was extracted with the RecoverAll Total Nucleic Acid Isolation Kit starting at the protease digestion step. Digestion was performed for 15 min at 50°C and 15 min at 80°C in the presence of RNasin. Extraction was performed according to manufacturer's instructions and the addition of RNAsin to all buffers just before use until final elution of RNA in DNAse-free water. Residual DNA was further digested using DNAse I (Invitrogen AM1906). RNAs were sorted at -80°C until further analysis. The libraries were prepared using the Illumina Stranded mRNA Prep Ligation Reference Guide, using 500-1000ng of total RNA.