Fig. 1

Workflow for genome-wide complementary mimic and inhibitor screens of host-encoded miRNAs impacting SARS-CoV-2 infection. (A) The miRNA mimic (1,239 targets) and inhibitor (1,255 targets) libraries were screened in duplicate in HeLa-ACE2 cells. Using high throughput liquid handling automation (Janus G3 robot), cells (800 per well) were reverse transfected using DharmaFECT 1 in 384 well plates at a final concentration of 25 nM. Using an EL406 liquid handling workstation, media was changed after 24 h. (B) At 72 h post transfection, plates were transferred to BSL-4, infected with SARS-CoV-2 (MOI 0.5, 24 h), and fixed for 1 h using 4% PFA. Plates were placed in polypropylene bags filled with 4% PFA and heat sealed prior to removal from the BSL-4 laboratory via a dunk tank containing MicroChemPlus (diluted 1/64). Plates were then transferred to a fume hood at BSL-3 and washed extensively with distilled water followed by PBS. Cells were stained with a rabbit polyclonal antibody targeting the SARS-CoV-2 N protein (Sino Biological, 40588-T62, Lot#HD14AP2604), and counter-stained with DAPI (nucleus), CellMask Deep Red (whole cell) and Phalloidin (actin cytoskeleton). (C) Plates were imaged using the CellInsight CX7 LZR High-Content Screening (HCS) Platform (16 fields/well) at the VCFG. High content image analysis was carried out using CellProfiler 3.0.0 image analysis software. Screen readouts included quantification of cell number, SARS-CoV-2 infection, cell cycle and morphology. Output data was analysed in R.