Lentiviral Production and Transduction of Expi293 Cells

cDNAs for soluble SARS-CoV-2 S and RBD (containing C-terminal His-tag) [11 (link)] were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene, #17452, gift from Eric Campeau & Paul Kaufman) using NEBuilder HiFi DNA Assembly Master Mix (NEB). Lentivirus was produced in HEK293FT by co-transfection of cDNA containing pLenti plasmids together with pCMV-dR8.2 dvpr (Addgene, #8455, gift from Bob Weinberg), pCMV-VSV-G (Addgene, #8454, gift from Bob Weinberg) and pAdVAntage (Promega) using FugeneHD (Promega). Supernatants were collected 48 hr post-transfection and filtered using a 0.45 μm syringe filter. For the transduction of Expi293 cells, five 6-well plates with 2 million cells per well were spin-infected with lentivirus diluted 1:2.5 in fresh Expi293 expression media under the following conditions: 2 hr, 33°C, 1,000 g. Cells were subsequently pooled together and cultured in 30 mL Expi293 expression media on a shaking incubator. 24 h post-transduction puromycin was added at 2 μg/mL and cells were selected for 7 days.