Single Nucleus Capture of Mouse Stria Vascularis

The method of adult mouse SV preparation has been previously described^{2 (link)}. Briefly, the lateral wall of the cochlea was microdissected from the bony wall of adult mouse cochlea and the pigmented strip in the cochlea lateral wall denoting the SV was microdissected from the spiral ligament using fine forceps. SV from all turns of the cochlea were collected. Samples were collected at the same time of day across individual mice and batches. For each collection, less than 1 h was spent prior to single nucleus capture on the 10 × Genomics Chromium platform. Sexes of mice were generally mixed for each experiment. 5 mice (2 female, 3 male P30 mice) were used for the methanol-fixed single nucleus capture and 6 mice (3 female, 3 male P30 mice) were used for the RNAlater-treated single nucleus capture. For the methanol-fixed sample, isolated cell nuclei obtained as previously described^{2 (link),23 (link),81 (link)}. Briefly, nuclei were suspended in 200 μL Dulbecco’s phosphate buffered saline (DPBS), then 800 μL of ice-cold methanol was slowly added drop-by-drop to the single nuclei suspension while gently stirring the nuclei suspension. Nuclei were moved to the freezer and incubated 30 min at − 20 °C. Subsequently, cells were rehydrated in wash and resuspension buffer (1 × PBS with 1% BSA and 0.2 U/ul RNase Inhibitor). Nuclei suspension underwent centrifugation (100 rcf, 5 min, 4 °C) and supernatant was removed and cells were resuspended in 50 μL of wash and resuspension buffer to obtain 700–1200 cells/μL prior to nuclei isolation and sequencing. For the RNAlater-treated sample, freshly dissected adult SV tissues were submerged in and stored in 0.7 mL of RNAlater solution (Catalog No. AM7020, ThermoFisher, Waltham, MA) at room temperature in a 1.5 mL Eppendorf tube and then stored at 4 °C overnight. After incubation, DPBS was added in equal volumes (0.7 mL) to the tube and gently mixed, then centrifuged at 500 g for 5 min at room temperature. Supernatant was removed and replaced with lysis buffer before previously described nuclei isolation and sequencing.