Puromycin medium

In total, 1.11 × 10⁵ cells in a volume of 1 ml were seeded in six-well dishes and incubated overnight at 37 °C, 5% CO₂. The virus stock was first diluted 1/40 followed by four five-fold serial dilutions in ice-cold medium containing 48 μg/ml polybrene (TR-1003-G, EMD Millipore) as the virus diluent (for a final concentration of 8 μg/ml). In total, 0.2 ml of each virus suspension was used to infect five wells, while, a sixth well was treated with 0.2 ml of virus diluent alone. Plates were returned to the incubator for 5 h incubation after which time each well was replenished to 2 ml with culture medium and incubated a further 48 h. Cells from each virus dilution infection well were harvested, and 10,000 cells in 100 μl were re-plated to a 96-well plate. Following overnight incubation, wells were treated with 12.5 μl of 72 μg/ml puromycin medium (A1113803, Thermofisher) (for a final concentration of 8 μg/ml), and replicate wells were treated with an equivalent volume of medium. After 72–96 h treatment, the proportion of infected, puromycin-resistant cells was determined by a viability assay using the CellTiter-Glo method, and infectious virus titer was calculated based on the virus volume used (see Supplementary Information Extended Methods).