A Manual Technique for Isolation and Single-Cell RNA Sequencing Analysis of Cochlear Hair Cells and Supporting Cells

Single-cell RNA sequencing is a powerful tool that can be used to characterize the transcriptional profile of low-abundance cell types in many organ systems; however, its application to the inner ear is especially challenging. Access to the membranous cochlea is hampered by the bony labyrinth, and the membranous labyrinth itself offers its own technical challenges due to tissue sparsity and difficulty in dissociating and isolating individual cells. Herein, we present a manual method to isolate individual inner hair cells, outer hair cells and Deiters’ cells from the murine cochlea at any postnatal time point. We describe the technique and downstream bioinformatic analysis and, by way of example, show how both short- and long-read single-cell RNA sequencing can be leveraged to profile transcript abundance and structure. This method has afforded us novel insights into the expression profiles of these specific cell types, revealing an unappreciated complexity in isoform variety in deafness-associated genes. Further application of this methodology may provide a more refined view of transcription in the organ of Corti, thereby improving our understanding of the biology of hearing and deafness.