Fibroblast cultures were grown on substrates coated with 0.1% gelatin (Millipore) in fibroblast media [DMEM (Invitrogen) containing 10% fetal bovine serum (FBS; Gemini Bio-products, Calabasas, CA, USA), non-essential amino acids, glutamate, and penicillin/streptomycin (Invitrogen)]. Lentiviral infection of human fibroblasts and conversion into induced neurons (iNs) were performed as described in33 with minor modifications. Briefly, miR-9/9*-124 together with NEUROD2, ASCL1 and MYT1L (kindly provided by the Crabtree lab, Stanford University) were expressed in human fibroblasts. Infected cells were then maintained in fibroblast media supplemented with 1 mM valproic acid (VPA, Sigma) for 3–4 days before selection with appropriate antibiotics in Neuronal Medium (NM, ScienCell, Carlsbad, CA, USA) supplemented with 1 mM VPA for 7 days. 3–4 days after antibiotic selection, cells were passaged onto poly-ornithine/laminin (20 µg/mL) coated 96-well plates and maintained in NM supplemented with 1 mM VPA for an additional 7 days, after which VPA was withdrawn for the remaining differentiation period. Media were changed every 3–4 days. 38–40 days after the initial lentiviral transduction, cells were treated with RNA Protect Cell Reagent (Qiagen, Valencia, CA, USA). Then, RNA extraction was performed using miRNeasy Micro Kit (Qiagen) as per manufacturer’s instructions.
