GSM5912098: 1-WT; Danio rerio; RNA-Seq
Sorted embryos were dechorionated with 1mg/ml pronase E and 1X HBSS and rinsed two twice with de-yolking solution (0.03mM MS-222 and 1X DMEM, high glucose and 1X B27), The embryos yolk was mechanically dissociated by gently pipetting the embryos in the de-yolking solution. Embryos were rinsed three times in de-yolking solution before micro dissecting the region of the otic vesicle and associated neurons with 0.33mm fine needles53. The isolated otic region was transferred with a fire-polished glass pipette in a 0.5ml Lo-bind tube coated with 10%BSA in 1X PBS90. Dissected tissue was kept on ice in de-yolking solution until cell dissociation. Tissue was washed with 1X HBSS, incubated at room temperature with 200µl of FACSmax (Gelantis) supplemented with 1 mg/ml of activated papain (Worthington) for 5 minutes and mechanically dissociated with 10%BSA/1XPBS coated tips 90. The dissociated cells were centrifuged at 300g for 5 minutes at 4°C. The dissociation solution was removed, and cells were washed twice with 1% BSA/1XPBS. Cells were counted using the Moxi Z automated cell counter (Orflo) using the Type S cassettes before the final spin. Cells were resuspended at 1200 cells/µl in 0.05%BSA/ 1X PBS and placed on ice. Single-cell cDNA libraries were generated using the Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10X Genomics) as suggested by manufacturer.