Single cell RNA-seq of in vivo cells

scRNA-seq for postnatal day 2 and day 7 mouse utricle was performed as follow: 3 mice (CD-1 background) were euthanized and their temporal bone removed. Utricles were harvested and incubated in thermolysin (Sigma-Aldrich) for 20 min at 37°C. Thermolysin was then replaced with Accutase (Sigma-Aldrich) and the tissue incubated for 3 min at 37°C followed by mechanical dissociation until a single cell suspension was obtained. After inactivation of the Accutase with 5% fetal bovine serum, the cell suspension was filter through a 35μm nylon mesh and processed for scRNA-seq. Dissociated cells were captured into a Chromium Controller (10x Genomics) for droplet-based molecular barcoding. Library preparation was performed using the 10x Single Cell Gene Expression Solution. Libraries from two utricular samples were sequenced across three lanes of an Illumina HiSeq4000 sequencer to produce paired-end 75 bp reads.

Initial scRNA-seq data processing, including demultiplexing, alignment to the mouse genome (mm10), and read counting were performed with cellranger (10X Genomics). The number of genes expressed, the number of UMIs detected, and the percentage of mitochondrial and ribosomal RNA were calculated for quality control. Cells with >5% of UMIs from mitochondrial genes were discarded. We applied the Seurat v3 Standard Workflow³⁷ to integrate cells across replicates, using 7,000 highly variable genes, 3000 anchors, and 50 dimensions. Subsequently, principal component analysis was performed, and cells clustered on a K-nearest neighbor graph based on Euclidean distance using the previously defined PCA dimensionality as the input. Cells were clustered using the Louvain algorithm to optimize the standard modularity function before performing dimensionality reduction via UMAP. Further analysis was performed by re-clustering selected sets of cells followed by differential gene expression analysis to identify unique cell markers.