Meme non essential amino acids

Lentiviral packaging of the pooled shRNA library was performed as previously described (Hoffman et al., 2014 (link)). For the viral packaging of individual shRNA constructs, 4 × 10⁶ 293T cells were seeded in a 100-mm poly-d-lysine–coated dish (Corning BioCoat; 356469) 1 d before transfection with 14 ml cell growth medium (DMEM, 10% FBS [631106; Clontech], 2 mM l-glutamine [25030; Invitrogen], 0.1 mM MEME nonessential amino acids [11140; Invitrogen], and 1 mM sodium pyruvate MEM [11360; Invitrogen]). For transfection, 7 µg packaging plasmid DNA (ViraPower lentiviral Packaging Mix, K497500; Thermo Fisher Scientific) was mixed with 5 µg expression construct DNA and 36 µl Fugene6 (E2691; Promega). OptiMEM (31985062; Thermo Fisher Scientific) was then added to the mixture for a total volume of 800 µl. 293T cells were incubated with the transfection reagent mixture for 24 h before the growth medium was refreshed. 72 h after transfection, virus was harvested and frozen for future experiments. For viral packaging of overexpression constructs (GFP and GFP-MCIDAS), the transfection procedure was performed as above, and then virus was concentrated with Lenti-X Concentrator (631231; Clontech) before freezing.