Amicon ultra 15 centrifugal ultrafilters

HEK 293T cells were maintained in high glucose DMEM supplemented with 10% fetal bovine serum (FBS). Cells were seeded in a 15 cm dish 1 day prior to transfection, such that they were 60–70% confluent at the time of transfection. For each 15 cm dish 36 μl of Lipofectamine 2000 (Life Technologies) was added to 1.5 ml of Opti-MEM (Life Technologies). Separately 3 μg of pMD2.G (Addgene #12259), 12 μg of pCMV delta R8.2 (Addgene #12263) and 9 μg of an individual vector or pooled vector library was added to 1.5 ml of Opti-MEM. After 5 minutes of incubation at room temperature, the Lipofectamine 2000 and DNA solutions were mixed and incubated at room temperature for 30 minutes. Medium in each 15 cm dish was replenished with 25 ml of fresh medium. After the incubation period, the mixture was added dropwise to each dish of HEK 293T cells. Supernatant containing the viral particles was harvested after 48 and 72 hours, filtered with 0.45 μm filters (Steriflip, Millipore), and further concentrated using Amicon Ultra-15 centrifugal ultrafilters with a 100,000 NMWL cutoff (Millipore) to a final volume of 600–800 μl, divided into aliquots and frozen at −80°C.