We utilized a mid-PRS line [line 420 (58)] iPSC sourced from the NIAAA/COGA Sharing Repository to generate excitatory-induced neurons (Ngn2-iNs), following the protocol as described in (57).In brief, when the iPSCs reached a confluency of approximately 70%, they were dissociated using Accutase and subsequently plated onto a six-well plate coated with Matrigel Matrix. These plated cells were then cultured in mTeSR plus medium, which contained RHO/ROCK pathway inhibitor Y-27632, doxycycline-inducible lentiviruses expressing transcription factors (Ngn2), and the reverse tetracycline-controlled transactivator (rtTA). The cells were cultured at 37°C with 5% CO2. After around 6 to 8 hours of lentivirus infection, the culture medium was replaced with Neurobasal medium (Gibco, catalog #21103049). This medium was supplemented with B-27 supplement (Gibco, catalog #17504044), l-glutamine (Gibco, catalog #35050061), and doxycycline (2 mg/ml; MP Biomedicals) to induce TetO expression.
