Amlodipine

Cellular cholesterol was measured using the Cholesterol-Glo assay (Promega). For each cell line (CRISPR-perturbed or drug-treated), we counted 100,000 cells, washed with 1xPBS twice and resuspended in 100 μL cell lysis solution for 30 min at 37°C. Following the incubation, 10 μL of cell lysate was used to measure the cholesterol levels in 96 well plates in a final reaction volume of 100uL per well (without esterase). Luminescence was measured after incubating the plate for 1 hour at room temperature in the dark. Using the same lysis sample, the protein concentration was determined using the BCA assay (Thermo). The cholesterol levels were normalized to the respective protein concentration of each sample. A549^ACE cells were incubated with 10 μM amlodipine (Sigma) or DMSO for 24 hours. Cholesterol levels were then determined as described above.

To identify druggable genes among the top CRISPR screen hits, we cross-referenced highly-ranked genes from the RRA analysis with the data table containing drugs and their gene targets was obtained from Drug Gene Interaction database (DGIdb, retrieved on June 3, 2020) as well as manual literature search (Cotto et al., 2018 (link)). Inhibitors that target the genes of interest (and remdesivir) was obtained from SelleckChem and MedChemExpress. The catalog number and vendor information is available in the Key Resource section. Amlodipine was obtained from Sigma (A5605).
To test drug efficacy in reducing SARS-CoV-2 infection, 10,000 A549^ACE2 cells were seeded per well of a 96-well plate. Cells were treated with inhibitors at 10 μM for two hours before infection and inhibitors were maintained throughout the course of infection. Cells were infected with SARS-CoV-2 at MOI of 0.1 for 36 hours and the cells were collected for analysis via qRT-PCR or processed for immunofluorescence (N protein and quantified by Celigo). For Figure 4E, the remdesivir data was collected in an independent experiment.