Lentivirus production and transduction

HEK 293 T cells were grown in 15 cm to ~50% confluence. For each plate, transfection was performed using Fugene6 (Promega), 6.2 μg of psPAX2, 0.620 μg pVSVg, 6.25 μg of CRISPR plasmid. After 30 min of incubation at room temperature, the mixture was added to the cells and incubated overnight. The next day harvest media was added (DMEM 30% FBS). After two collections at 24 h each, the harvested virus was passed through a 0.45 μm filter. Transductions were performed by seeding cells at ~40% confluence into six-well dishes, then the following day adding 0.5 mL of virus, 0.5 mL of media, and 2 μL polybrene to the cells. The cells were then centrifuged to the following specifications: 1 h, 4 °C, 2500 RPM. Following the spin, fresh media without virus or polybrene was placed onto the cells. The following day, cells were selected with appropriate selection antibiotic.