Single-Cell Transcriptomics of Fish Brains

Wild-type fish were processed for 10× Genomics single-cell transcriptome sequencing. Whole brains or different brain regions (Fore, OT, Hind, and sub-OT) were dissected as anatomical structures (Figure 1B, Figure 1—figure supplement 1A). Tissues were dissociated with 300 μL papain (28 units/mL, Worthington) in papain solution (1% DNase, 12 mg/mL L-cysteine in DMEM/F12), incubated at 37°C for 15 min with proper mix methods (Yu and He, 2019 (link)). Dissociated cells were washed twice with washing buffer (100 mL: 650 μL 45% glucose, 500 μL 1 M HEPES and 5 mL FBS into 93.85 mL 1× DPBS, sterilized with a 0.22 μm pore size filter), and sterilized with 40 μm cell strainers (BD Falcon) into 300–400 μL washing buffer.