title: GSM6500163: 14dpf_Sox10CreUbiswitch_ear; Danio rerio; RNA-Seq
library_construction_protocol: For 14 dpf animals, heads from converted Sox10:Cre; ubb:LOXP-EGFP-LOXP-mCherry fish were decapitated at the level of the pectoral fin with eyes and brains removed. For 12 mpf animals, utricle, saccule, and lagena were extracted from converted Sox10:Cre; ubb:LOXP-EGFP-LOXP-mCherry fish after brains and otolith crystals were removed. Dissected heads and otic sensory patches were then incubated in fresh Ringer's solution for 5–10 min, followed by mechanical and enzymatic dissociation by pipetting every 5 min in protease solution (0.25% trypsin (Life Technologies, 15090-046), 1 mM EDTA, and 400 mg/mL Collagenase D (Sigma, 11088882001) in PBS) and incubated at 28.5 °C for 20–30 min or until full dissociation. Reaction was stopped by adding 6× stop solution (6 mM CaCl2 and 30% fetal bovine serum (FBS) in PBS). Cells were pelleted (376 × g, 5 min, 4 °C) and resuspended in suspension media (1% FBS, 0.8 mM CaCl2, 50 U/mL penicillin, and 0.05 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO) in phenol red-free Leibovitz's L15 medium (Life Technologies)) twice. Final volumes of 500 μL resuspended cells were placed on ice and fluorescence-activated cell sorted (FACS) to isolate live cells that excluded the nuclear stain DAPI 10X Genomics Chromium Single Cell 3′ Library and Gel Bead Kit v.3.1 (14 dpf) or Single Cell Multiome ATAC + Gene Expression kit (12 mpf)