Suzanne de Bruijn

298 Chapter 6 2. The replacement of bulk-sequencing with single-cell approaches Both the inner ear and the retina are considered highly complex tissues consisting of a variety of highly specialized cells. Spatiotemporal differences in gene expression (levels) between and within the distinct cell types allow the coordination of various functions that enable hearing or vison. Each cell type expresses many context-specific transcripts and splice variants and thus protein isoforms that all contribute to the overall gene expression profile. In traditional expression studies, the transcriptome or proteome of the retina or inner ear is analyzed in bulk; mRNAor protein content of the complete tissue is pooled and analyzed together. 92 Gene expression changes that can be attributed to only small subsets of cells are easily missed. This severely complicates the analysis of expression profiles between two conditions such as affected and non-affected tissues. Single-cell approaches such as single-cell RNA sequencing (scRNA-seq) addresses this limitation by investigating the transcriptome of individual cells in a parallelized, high- throughput fashion . Cell sorting-based approaches allow capturing of the mRNA profile of hundreds to thousands of selected cells simultaneously. Expression profiles of single cells can be directly compared, and clustered to discriminate cell populations. scRNA- seq has been successfully performed to study both ocular 93 and inner ear 62 tissues. The approach allowed identification of cellular subclasses within cell types that were previously considered identical. For instance, 21 distinct clusters of amacrine cells could be distinguished in an scRNA-seq study in the mouse neural retina. 94 Important and significant differences could be observed by performing spatial and temporal expression analysis between foveal and peripheral retinal gene expression. Also in the inner ear, scRNA-seq data identified novel cellular subtypes: three subtypes of murine spiral ganglion neurons type I could be found. 95,96 scRNA-seq approaches can be applied to study retinal and inner ear disease, and can be expected to replace bulk-sequencing approaches in the future. Unannotated and alternatively splice transcripts in specialized cell types can be revealed, that may contain novel exons or have a regulatory function. Pathogenic variants in one of these previously unannotated exons can be easily overlooked or may have been wrongly deemed intronic and non-causative in the past. Both the inner ear and the retina express tissue-specific splicing factors that produce a large number of alternatively-spliced transcripts, 97,98 which are still poorly characterized. scRNA-sequencing approaches are necessary to close this knowledge gap, and are required to interpret and complete our understanding of (potentially) disease-associated variants. Ray et al. stated that thousands of mRNAs are present in the retina, including the previously overlooked CRB1