Production of Lentiviral Particles Using HEK293T Cells and Human Kinome CRISPR Knockout Library

HEK293T cells at 80-90% confluency were co-transfected with the Human kinome CRISPR knockout library (Brunello, Addgene Cat# 1000000083), psPax2 (Addgene Cat# 12260, RRID: Addgene_12260) and pMD2.G (Addgene Cat# 12259, RRID: Addgene_12259) at a ratio of 7:5:2 using Neofect DNA transfection reagent (Neofect, TF201201). After 16-20 hours, the culture media was replaced with viral production medium (Lonza). The viral supernatant was collected after 48 hours, spun at 4°C (3000 rpm) for 10 minutes, filtered with a 0.45 µm filter (Millipore), and concentrated by centrifugation at 120,000 g in a Beckman Ultracentrifuge (Optima L-100XP). The virus was then aliquoted and frozen at -80°C.