figshare
Browse
GenomeWideSiRNA_SARSCoV2.xlsx (4.05 MB)

Genome-wide siRNA screen in Caco2 cells infected with SARS-CoV-2

Download (4.05 MB) This item is shared privately
dataset
modified on 2021-08-19, 23:54

A genome-wide siRNA screen was carried out in human Caco2 cells to identify host factors that affect the replication of SARS-CoV-2.


Screen. The whole-genome siRNA library ON-TARGETplus SMARTpool (Dharmacon) was arrayed in 384-well plates (Greiner) at a concentration of 0.5 pmol per well. In addition, non-targeting siRNAs (scrambled) were added to each plate as negative controls, and siRNAs targeting SARS-CoV-2 entry factors ACE2 and TMPRSS2 were included as positive controls. siRNAs were mixed with 0.1 ml Lipofectamine RNAiMAX transfection reagent diluted in 9.90 ml Opti-MEM media (both reagents from Thermo Fisher Scientific), followed by addition of 3,000 Caco2 cells diluted in 40 ml EMEM media (Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Gibco), and 50 U/mL penicillin - 50 µg/mL streptomycin (Fisher Scientific). After a 48 h incubation period at 37°C, 5% CO2, cells were infected with SARS-CoV-2 (USA-WA1/2020) at a multiplicity of infection (MOI) of 0.625 for additional 48 h at 37°C, 5% CO2. Cells were then fixed with 4% PFA (Boston BioProducts) diluted in PBS for 4 h at room temperature, then washed twice with PBS and permeabilized with 0.4% Triton X-100 for 15 min at room temperature. Cells were blocked with 3% BSA (Sigma) for 30 min at room temperature, followed by an overnight incubation at 4°C with a primary antibody against SARS-CoV-2 nucleoprotein (N) protein diluted 1:3,000 in 3% BSA. Following three washes with PBS, cells were incubated for 1 h at room temperature with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific) diluted 1:3,000 in 3% BSA. Following three washes with PBS, cells were stained with DAPI (4,6-diamidine-2-phenylindole, KPL), and plates were sealed and stored at 4°C until imaging.

High-content imaging and data analysis. SARS-CoV-2 replication after each individual target knockdown was quantified using high-content imaging. The assay plates were imaged with the Opera Phenix imaging system located at the Conrad Prebys Center for Chemical Genomics (CPCCG) and analyzed using the analysis software Columbus v2.5 (Perkin Elmer). Based on the number of Alexa 488+ objects and the number of DAPI+ objects, the % of infected cells was quantified. The log2FC infection was calculated relative to the median of each plate. Cytotoxicity resulting from siRNA transfection was evaluated by normalizing the % of DAPI+ objects to that of the negative control scrambled siRNAs.

Funding

U19 AI135972

U19 AI118610