Mgoac

The 40μL reactions contained 50% HEK293T translation extract, 2mM ATP, 0.05mM GTP, 7mM tris(2-carboxyethyl)phosphine, 28mM HEPES pH 7.5, 2mM creatine phosphate (Roche), 0.01μg/μl creatine kinase (Roche), 2mM Mg(OAc)₂ and 10μM of the amino acid mixture (Promega). Reactions were incubated with 5μM of the indicated nsp1 protein for 30 min on ice. 5μM of 5′-Guanylyl imidodiphosphate (GMPPNP) (Sigma-Aldrich) and 500μM of cycloheximide (CHX) (Sigma-Aldrich) was then added and incubated at RT for 10 min, whereupon 1μg of the HBB-nLuc mRNA was added and reactions were incubated at 30°C for 15 min. Reactions were stopped with the addition of 400μL of RNase free water and equal amounts of TRIzol reagent (ThermoFisher). RNA was extracted and resuspended in RNase free water then added to primer extension buffer containing 7mM MgOAc (Sigma-Aldrich), 100mM KOAc (Sigma-Aldrich), 50mM Tris-HCl, 1mM DTT, 1 μg/μL of SuperScript IV RT (Ambion), 550μM of DNTP mixture (ThermoFisher) and 500nM of Cy5 labeled primer (IDT). The reaction was incubated at 30°C for 15 minutes then ethanol precipitated. DNA pellets were washed twice with 70% ethanol, dried, and resuspended in 95% formamide solution containing 10mM EDTA. Samples were loaded onto a pre-run 6% UREA-PAGE gel then imaged using an Amersham Typhoon 5 laser-scanner (Cytiva).