DRG neuronal culture and transfection

Primary DRG neuronal cultures and viral transfection were carried out as described.¹³ Briefly, adult mice were euthanized with isoflurane and all DRG were collected in cold Neurobasal Medium (Gibco/ThermoFisher Scientific) with 10% fetal bovine serum (JR Scientific, Woodland, CA), 100 units/ml Penicillin, 100 µg/ml Streptomycin (Quality Biological, Gaithersburg, MD) and then treated with enzyme solution (5 mg/ml dispase, 1 mg/ml collagenase type I in Hanks’ balanced salt solution (HBSS) without Ca²⁺ and Mg²⁺ (Gibco/ThermoFisher Scientific)). After trituration and centrifugation, dissociated cells were resuspended in mixed Neurobasal Medium and plated in a six-well plate coated with 50 µg/ml poly-D-lysine (Sigma, St. Louis, MO). The cells were incubated at 95% O₂, 5% CO₂, and 37℃. One day later, 0.5μl of virus (titer ≥ 1 × 10¹²/ml) was added to each 2-ml well. Neurons were collected two to three days later.