Prior to beginning, NPC medium was pre-warmed in a 37 °C water bath. Lentiviral aliquots of CMV-rtTA (Addgene ID: 19780), TetO-mNgn2-P2A-PuroR (Addgene ID: 52047), TetO-eGFP (Addgene ID: 30130), TetO-eGFP-PuroR (Addgene ID: 19780) and TetO-PuroR were thawed at room temperature and added to NPC medium. Lentiviral mNgn2 transduction was achieved with either CMV-rtTA, TetO-mNgn2-P2A-PuroR and TetO-eGFP or, CMV-rtTA and TetO-mNgn2-P2A-PuroR transduction. As a control, either CMV-rtTA and TetO-eGFP-T2A-PuroR or CMV-rtTA and TetO-PuroR were used. The amount of lentivirus added was adjusted to achieve multiplicity of infection of 1–10.
