Lentivirus production and transduction

For lentivirus production, desired transfer plasmid (12 μg) was cotransfected with 3 μg of VSV-G coat protein vector, pMD2.G (Addgene, 12259), and 6 μg of psPAX2 (Addgene, 12260) in HEK293T cells using Lipofectamine 3000 reagent in 10-cm plate. After overnight transfection, fresh medium was replenished, and 24 hours later, the first batch of viral supernatant was collected and stored in 4°C. Cells were replenished with fresh medium again, and the final batch of lentivirus supernatant was collected 24 hours later. Both the viral batches were pooled and filtered through 0.45-μm surfactant-free cellulose acetate membrane filter. As needed, lentiviral particles were either stored in −80°C or used immediately for transduction. For transducing adherent cells, 1 × 10⁵ cells were seeded a day before transduction. Cells were incubated with lentiviral particles with polybrene (6 μg/ml) for 6 hours. Cells were then replenished with fresh medium and grown for 24 hours followed by appropriate antibiotic selection. To transduce suspension cells, 8 × 10⁵ cells per well of a 12-well plate were spinoculated with lentiviral particles with polybrene (6 μg/ml) at 800g for 1 hour at 32°C. Lentiviral supernatants were discarded as per Northwestern University Biological Safety manual, and cells were replenished with fresh medium. Remaining steps were the same as those of adherent cells.