On day zero plate 10 million 293T cells in 30 milliliters of antibiotic-free DMEM containing 10%FBS in a 15 centimeter dish. Prepare a total of 10 such dishes. On the next day, which is day one, confirm that the cells are 80%confluent and ready for transfection.
Then in a 50 milliliter conical tube sequentially, add 600 microliters of 0.5 micrograms per microliter packaging plasmid mix and 60 microliters plasmid barcode library. In a separate 15 milliliter conical tube mix 900 microliters of transfection reagent and 12 milliliters DMEM by vortexing. Add 12.9 milliliters of this mixture to the DNA mix and just flick to combine.
Without mixing any further, incubate the mix at room temperature for 15 minutes. Then drop wise add 2.5 milliliters of the incubated mixture to each 15 centimeter dish containing the 293T cells and incubate the cells overnight in a tissue culture incubator. On day three, harvest the virus by passing the media through a 0.2 micron filtration unit.
Aliquot the filtrate containing viral particles into 15 milliliter conical tubes. Additionally, prepare five one milliliter aliquots of filtered virus in cryo vials for viral titering. To titer the lentiviral pools, add 65 microliters of Cationic polymer to a sterile glass flask containing 65 milliliters of tumor cell growth media.
Pipette one milliliter of the polymer containing media per well into 11 six-well tissue culture plates. Next trypsinize the early passage tumor cells. After counting the cells using a preferred method, transfer the cells into the six-well plates.
Thaw the one milliliter aliquots of the 48 hour lentivirus from the freezer in a water bath at 37 degrees Celsius. Follow the table to prepare the infection for each viral module.
