Pierce gaussia luciferase glow assay kit

Seven days after transfection with rabbit HEV-3ra LRGluc indicator replicon, the cell culture medium (100 μL) was removed from a 96-well plate for analysis of extracellular luciferase expression activity. Cell culture media were collected at different time points to monitor the kinetic changes in luciferase expression activities. Luciferase activity was measured with the Pierce Gaussia Luciferase Glow assay kit (Thermo Scientific, Waltham, MA, USA). Briefly, 20 μL/well cell culture medium or cell lysate was added to each well of a black, opaque 96-well plate, followed by the addition of 50 μL Gaussia substrate. After 10 min incubation for signal stabilization, the luminescence was detected and recorded using a GloMax Discover microplate reader (Promega, Madison, WI, USA). Each LRG mutant replicon experiment was performed in quadruplicate. Luminescence-based antiviral assay for HEV was performed with ribavirin (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1 μM or 10 μM following an established protocol (59 (link)).

A custom siRNA library targeting each individual RTC-proximal factor (On Target Plus, SMART pool, 96-well plate format, Dharmacon, GE Healthcare) was ordered. Additionally, a deconvolved library of 4 individual siRNAs was purchased for selected targets. 10 nM siRNA were reverse transfected into L929 cells (8*10³ cells per well) using Viromer Green (Lipocalyx) according to the manufacturer’s protocol. Cells were incubated 48 hr at 37°C 5% CO₂ and cell viability was assessed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega). Cells were infected with MHV-Gluc (MOI = 0.05, 1000 plaque forming units/well), washed with PBS 3 h.p.i. and incubated in MEM+/+for additional 9 or 12 hr. Gaussia luciferase was measured from the supernatant using Pierce Gaussia Luciferase Glow Assay Kit (ThermoFisher Scientific). Experiments were carried out in four independent replicates and both cytotoxicity values and luciferase counts were normalized to the corresponding non-targeting scrambled control of each plate. A one-way ANOVA (Kruskal-Wallis test, uncorrected Dunn’s test) was used to test the statistical significance of reduced viral replication (mean <95% as compared to scramble control, n = 216). The R package ggplot2 was used to create the bubble plot (Figure 4b).