Additional file 1 of Single-cell RNA-sequencing of stria vascularis cells in the adult Slc26a4-/- mouse
Creators: name: Koh, Jin-Young
name: Affortit, Corentin
name: Ranum, Paul T.
name: West, Cody
name: Walls, William D.
name: Yoshimura, Hidekane
name: Shao, Jian Q.
name: Mostaert, Brian
name: Smith, Richard J.H.
Publisher: name: figshare
Year: 2023
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1 Workflow. Single cells from the stria vascularis (SV) were harvested manually from P30 Slc26a4+/+, Slc26a4+/−, and Slc26a4−/− mice as a source of full-length RNA, which was used to prepare a library for single-cell RNA sequencing. Data analysis using uniform manifold approximation and projection (UMAP) identified four types of cochlear SV cells. Cluster-defining genes from unbiased clustering analysis was used to examine gene enrichment. In addition, we used clustering-defining genes to filter each cell type. Finally, we defined pH-dependent differential expressed genes in Slc26a4−/− as compared to Slc26a4+/+ in a cell-type- specific manner. Fig. S2. (Related to Fig. 1): SV Cells and size measurement (A-D) Representative images of isolated cells for the four-cell type cluster. Measure the cell’s perimeter as a region of interest (ROI). Scale bar: 10 μm. (IMC, intermediate cell; MC, marginal cell; RC, root cell; SC, spindle cell). (A) IMCs of the cochlear SV. The median perimeter was 59.80 ± 1.42 μm (n = 30). (B) SCs of the cochlear SV. The median perimeter was 64.49 ± 1.73 μm (n = 19). (C) RCs of the cochlear SV. The median perimeter was 65.38 ± 2.34 μm (n = 11). (D) MCs of the cochlear SV. The median perimeter was 65.79 ± 1.29 μm (n = 16). (E) Box plots of the radius of each cell. Y-axis is the radius of cells *, p &lt; 0.05. The radius of IMC (9.52 ± 0.23 μm) is significantly smaller than the radius of SC (10.26 ± 0.28 μm; p = 0.043) and MC (10.47 ± 0.21 μm; p = 0.0033). There is no difference in the radius of IMC and RC (p = 0.2). There is no difference in the radius of SC and RC (p = 0.76). There is no difference in the radius of SC and MC (p = 0. 55). There is no difference in the radius of RC and MC (p = 0.58). Each dot represents a single cell. Fig. S3. (related to Fig. 1) Quality control and principal components (PCs) of the SV cells. (A) PCs are shown as solid-colored curves. There is a sharp drop-off in p-values after the first 8 PCs. A dashed line is a uniform distribution. (B) A ranking of principal components (PCs) based on the percentage of variance. The black dots are 20 different PCs. Fig. S4. (Related to Fig. 3): Representative confocal images showing the cellular localization of pendrin and carbonic anhydrases in the inner ear (A-B) and kidney (C-D). (A-B) Immunostaining of pendrin (green) and CA13 (red) nuclei (blue), and F-actin (grey). Scale bar: 20 μm. Note: In Slc26a4−/− mice, pendrin and CA13 staining are negative. (C-D) Cryosections of the mouse kidney tissue are shown as positive and negative expression controls. Scale bar: 50 μm. Note: In Slc26a4+/+ mice (C), pendrin (green) and CA2 (red) co-localize in the intercalated cells. However, in Slc26a4−/− mice (D), pendrin and CA2 staining are negative