Vero-E6 were grown in DMEM+10% FBS+1% PBS and incubated at 37°C/5% CO2. Approximately 7.5x105 Vero-E6 cells were plated per well in a 6-well plate prior to transfection. LNAs were transfected at a final concentration of 400nM per well using the TransIT-Oligo reagent, including a reagent only transfection control (Mirus, MIR 2164). One day post-transfection, transfected or control Vero-E6 cells were plated at 2.5 × 103 cells per well in a 384-well plate in phenol free media and were then infected with icSARS-CoV-2mNG at a MOI of 1.0 (Xie et al., 2020)

Infected cell frequencies, as quantified by mNeonGreen expression, were assessed at 24 hours post-infection by high content imaging (Cytation 5, BioTek) configured with bright field and GFP cubes. Total cell numbers were determined from bright field images using Gen5 software. Object analysis measured the number of mNeonGreen positive cells. Percent infection was calculated as the ratio between the total number of mNeonGreen+ cells and total cells.

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