Screening Ubiquitination Machinery in TMPRSS2-HiBiT Cells

TMPRSS2-HiBiT cells were screened with an siRNA library targeting Ubiquitination-related machinery^{47 (link)}. Briefly, 25 ng of siRNA was mixed with Lullaby transfection reagent (OzBiosciences) and diluted in Opti-MEM media. The transfection mixture incubated at room temperature for 20 min, and was added to 20 μL of HITES + 10%FBS media with 2000 cells. Following 72 h knockdown, Lytic HiBiT luciferase assays were performed using manufacturer’s protocol. For conformational specific gene silencing, small interfering RNAs were selected and purchased from IDT, and transfected in cells using Lullaby siRNA transfection reagent, with Negative Control DsiRNA transfected as control. Subsequent analysis was performed after 72 h of knockdown.