Characterisation of protease activity duringSARS-CoV-2 infection identifies novel viralcleavage sites and cellular targets withtherapeutic potential

Virus infections in siRNA-based cellular protein knockdowns - imaging dataset for cell viability
Host proteins were knocked-down in A549-Ace2 cells using specific dsiRNAs from IDT. Briefly, A549-Ace2 cells seeded at 1x10$^{4}$ cells/well in 96-well plates. After 24 hours, each well was transfected with 5 pmol of individual dsiRNAs using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. 24 hours post transfection, the cell culture supernatant was removed and replaced with virus inoculum (MOI of 0.1 PFU/cell). Following a 1 hour adsorption at 37°C, the virus inoculum was removed and replaced with fresh 2\% FBS/DMEM media. Cells were incubated at 37°C for 3 days before supernatants were harvested. Samples were either heat-inactivated at 80°C for 20 min and viral RNA was quantified by RT-qPCR, using previously published SARS-CoV-2 specific primers targeting the N gene \cite{ChuClinChem}. RT-qPCR was performed using the Luna Universal One-Step RT-qPCR Kit (NEB) in an Applied Biosystems QuantStudio 7 thermocycler, using the following cycling conditions: 55 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 sec, followed by 60 °C for 1 min. The quantity of viral genomes is expressed as PFU equivalents, and was calculated by performing a standard curve with RNA derived from a viral stock with a known viral titer. Alternatively, infectious virus titers were quantified using plaque assays as described above.

To quantify siRNA-based cellular protein knockdowns, A549-Ace2 cells were seeded and transfected with individual dsiRNAs as described above. After 24 hours incubation at 37 °C cells were lysed and RNA was extracted using Trizol (ThermoFisher Scientific) followed by purification using the Direct-zol-96 RNA extraction kit (Zymo) following the manufacturer’s instructions. RNA levels of target proteins were subsequently quantified by using RT-with the Luna Universal One-Step RT-qPCR Kit (NEB) in an Applied Biosystems QuantStudio 7 thermocycler using gene-specific primers. Expression levels were compared to scrambled dsiRNA-transfected cells und normalized to expression of human beta-actin. Knockdown efficiencies were calculated using ΔΔCt in Matlab. 

To assess cell viability after siRNA knockdowns, cells were seeded and transfected as described above. 24 hours after transfection cell viability was measured using alamarBlue reagent (ThermoFisher Scientific),  media was removed and replaced with alamarBlue and incubated for 1h at 37 °C and fluorescence measured in a Tecan Infinite M200 Pro plate reader. Percentage viability was calculated relative to untreated cells (100\% viability) and cells lysed with 20\% ethanol (0\% viability), included in each plate.

For cell counting to determine cell numbers, cells were fixed in formalin to deactivate virus. The fixed cells were stained with 5µg/ml of Hoechst 33258 (Sigma). The assay plates were imaged on an IX-83 automated inverted microscope (Olympus) using a 10x objective. The DAPI settings (Ex UV 377/50, Em 415–480) were used to image Hoechst 33258. The acquisition setup was configured to image 4 sites per well. The nuclei were identified using the object detection module in the ScanR analysis software.

Data provided in two zip files. Sample layout/key within the plates is provided within the zip files.