Lentiviral Transduction of Glioma Stem Cells

The sgRNA library and single-targeted sgRNA lentiviral plasmids for GSC transduction were purchased from Addgene (#73179 and #52961, respectively). Lentiviral particles were generated as previously described^{67 (link)}. For lentiviral transduction, GSC tumorspheres were dissociated into single cells using Accutase (Innovative Cell Technologies), resuspended in GSC media, and optimal lentivirus was added in order to achieve 30–40% infection efficiency. GSCs were then washed once after 12 h, resuspended in fresh GSC media, and cultured for 2 days. Transduced cells were enriched by puromycin (Thermo Fisher Scientific, final concentration 1 μg/ml) selection for seven continuous days.