Transfection and lentivirus production/infection

Cells were plated on 100-mm dishes 24 h before the experiment, and cloned circRNAs plasmids were transfected with Lipofectamine Stem Reagent (STEM00003, Thermo Fisher Scientific, Waltham, MA, USA) following the company guidelines. The empty pcDNA3.1(+) CircRNA Mini Vector was used as a control. For pLVX-IRES-ZsGreen1, pLVX-shRNA2 constructs and glutamatergic differentiation vectors (TetOhNGN2-P2A-EGFP-T2A-PuroR and CMV-rtTA), lentiviral particles were produced in HEK293T cells as described below. HEK293T cells were cultivated in DMEM with GlutaMAX (31966-021, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (10270, Thermo Fisher Scientific, Waltham, MA, USA) and grown at 37°C in a humidified atmosphere of 5% CO₂. HEK293T cells were triple-transfected with specific vectors plus packaging plasmids with jetPRIME (114-15, Polyplus-transfection, Strasbourg, France) according to the manufacturer’s recommendations. Viral particles with pLVX-IRES-ZsGreen1 and pLVX-shRNA2 empty vector were used as a control. Lenti-X Concentration (PT4421-2, Clontech, Mountain View, CA, USA) was used for concentrating viral particles in DMEM-F12 medium. Cells were infected with viral particles, followed by a 2-day recovery period before the experiments started. ZsGreen1 was used as a marker to visualize transductants cells by fluorescence microscopy for pLVX-IRES-ZsGreen1 plasmids, whereas the TetO-hNGN2-P2A-EGFP-T2A-PuroR vector was selected by puromycin (ant-pr, InvivoGen, San Diego, CA, USA) and visualized with EGFP fluorescence.