Primary neuronal culture and lentivirus transduction

ICR pregnant females were deeply anesthetized by isoflurane inhalation, and a small incision was made in the abdominal wall to harvest their embryos for brains. Cortical tissue from E16.5 ICR embryonic brains was isolated and dissociated to acquire cortical neurons using the Worthington Papain Dissociation kit according to manufacturer’s protocol (Worthington Inc.). Cortices from several embryonic brains were pooled for plating. Neurons were plated at 100,000 cells per well of a 6-well plate. Cultures were maintained in a regularly replaced Neurobasal media (Invitrogen, Grand Island, NY) with B27 supplement (Invitrogen), penicillin-streptomycin supplement (Life Technologies) and L-glutamine (Invitrogen)⁹⁴.

Neurons were incubated at 37 °C and 5% humidity. On 7^th day-in-vitro (DIV7), control (VSM5954, Dharmacon; GE Healthcare) and MiR142-3p-OE (VSM6215-213652311, Dharmacon, Lafayette, CO) lentiviruses were added at the titer concentration of 10⁸ transfection units/ml. On DIV11, total RNA was extracted using miRVana RNA isolation kit. Two independent experiments were performed from two different pregnant females and each experiment was conducted with three replicates.