Three days before lentiviral transduction, iPSC-derived NPCs (WTiNPCs and P53KDiNPCs, passages 6–8) were passaged. On the day of transduction, each of the four lentiviral preparations (RasV12, EGFR D770-N771; EGFR (del3) and src Y527F) was thawed on a bed of ice, mixed at a 1:1:1:1 ratio (MOI of 0.75 per virus) in NSMM medium in the presence of polybrene (4 μg ml−1). NPCs culture medium was replaced with the lentiviruses containing medium and cells were incubated overnight at 37 °C. The next day, the medium was replaced with fresh NSMM. Transduced iNPCs (Ras/EGFR/SrciNPCs, P53KD-Ras/EGFR/SrciNPCs were passaged for at least two more passages for stabilization before performing analyses.
