Arabidopsis seeds of wild-type Col-0 were sterilized with 75% ethanol and then germinated vertically and grown on 1/2 Murashige & Skoog basal (MS) medium without sucrose at 22°C under dark conditions for 9 d. The hypocotyl explants (~1 cm in length) were cut and cultured on CIM (MS basal medium with vitamins, 2% w/v sucrose, 11 μM 2,4-dichlorophenoxyacetic acid, 0.2 μM kinetin) under 24-h light conditions at 22°C for 6 d. Protoplasts were isolated by enzyme digestion method. Single-cell RNA-seq were performed using BD Rhapsody system.