Primary Cortical Neuron Culture and Cloning

Primary cortical cultures were prepared from postnatal mouse pups. As previously described^{37 (link)}, the cortex was cut into small pieces and digested at 37 °C for 15 min. To stop digestion, the complete medium (DMEM supplemented with 10% FBS) was applied. After digestion, the tissue suspension was filtered with a 40-µm cell strainer (Falcon) and centrifuged at 1000×g for 8 min. Cells were counted with cell counting equipment and seeded in 24-well plate at 37 °C with 5% CO₂. After 4-h incubation, the culture medium was removed and replaced with maintenance medium, containing Neurobasal (Gibco), B27 (Invitrogen), and glutamine (Invitrogen). Anti-Tuj-1 (1;1000 mouse, Beyotime, China) immunostaining was used for characterization of the cultured neurons. HEK293, U251, and SH-SY5Y cells were grown in DMEM supplemented with 10% FBS at 37 °C with 5% CO₂. For selecting the single clone cells, lentiviral infected cells were placed in 96-well plate by glass micropipette (Sutter Instrument). The genotypes of single clone cell lines were characterized by PCR and sanger sequencing.