GSM7817937: A549-ACE2_bystander_total_cov_depleted_2; 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. 500-1000 ng of total RNA were depleted of SARS-CoV-2 RNA using custom designed probes. The probes were synthesized using the NC_045512.2 Wuhan-Hu-1 complete genome reference. The design was made by Illumina and is composed of 459 probes, separated into two pools synthetized by IDT. For the SARS-CoV-2 depletion, we mixed both pools and used 1µl of this mix per sample, replacing the Ribozero+ probes at the ribodepletion reaction step of the Illumina Stranded Total RNA prep ligation protocol. The SARS-CoV-2 depleted RNA samples were normalized to 300ng and ERCC Spike was added as recommended by the protocol ERCC RNA Spike-In Control mixes User Guide. The libraries were prepared using the Illumina Stranded mRNA Prep Ligation Reference Guide.