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Comprehensive characterization of transcript isoform in mouse retina with ONT long read single-cell RNA sequencing

ABSTRACT: The retina, a complex neural tissue, encompasses a vast diversity of over 100 distinct cell types, each characterized by unique features such as morphology, function, location, and transcriptomic profiles. However, the extent of mRNA alternative splicing within individual cell types in the retina remains largely uncharted territory. To bridge this knowledge gap, we employed single-cell RNA sequencing, utilizing both short-read and long-read high-throughput technologies. Our comprehensive analysis profiled the transcriptomes of 29,191 mouse retina cells, resulting in a dataset comprising 1.54 billion Illumina short reads and 1.40 billion Oxford Nanopore long reads. Remarkably, this exploration unveiled a staggering 44,325 transcript isoforms, with 38% of them representing entirely novel discoveries and 17% exhibiting cell-class-specific expression patterns. Intriguingly, while many isoforms were expressed across various cell types, their distribution often displayed variability among them. This research substantially enriches the catalog of transcript isoforms, laying the groundwork for further investigations into the role of alternative splicing in retinal biology and its implications for related diseases. It represents a crucial step toward unraveling the intricacies of retinal function and offers promising insights into the molecular mechanisms underlying retinal disorders.

ORGANISM(S): Mus musculus

PROVIDER: GSE255520 | GEO | 2024/11/04

REPOSITORIES: GEO

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Project description:Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes. Reprinted from <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=25446321">Whitmore, S.S., Wagner, A.H., DeLuca, A.P., Drack, A.V., Stone, E.M., Tucker, B.A., Zeng, S., Braun, T.A., Mullins, R.F., Scheetz, T.E., 2014. Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq. Experimental Eye Research</a>. Used under <a href="http://creativecommons.org/licenses/by-nc-sa/3.0/">Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported </a>license. Additional RNA sequencing was performed on temporal, macular, nasal, inferior, and superior retina from a fifth subject and is included in this dataset. See original publication for details.

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Comprehensive characterization of transcript isoform in mouse retina with ONT long read single-cell RNA sequencing

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