Abstract: FR-PO0342
Single-Nucleus Long-Read RNA Sequencing Unravels Isoform-Level Regulation of Gene Expression in Normal and Acutely Injured Proximal Tubule Cells
Session Information
- Top Trainee Posters - 2
October 23, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 01:06 PM - 01:12 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Oda, Yasuhiro, Washington University in St Louis, St. Louis, Missouri, United States
- Noonan, Megan L., Washington University in St Louis, St. Louis, Missouri, United States
- Dixon, Eryn E., Washington University in St Louis, St. Louis, Missouri, United States
- Wu, Haojia, Washington University in St Louis, St. Louis, Missouri, United States
- Humphreys, Benjamin D., Washington University in St Louis, St. Louis, Missouri, United States
Background
Single-cell short-read RNA-seq has elucidated transcriptomic changes across kidney cell types at the gene expression level. However, its limited read length precludes reliable isoform identification. We explored alternative splicing regulation in healthy and AKI murine kidneys by leveraging long-read sequencing with single-cell and single-nucleus RNA-seq technologies.
Methods
Single-cell and single-nucleus long-read RNA-seq datasets were generated from kidneys of three adult male C57BL/6 mice. An additional three adult male C57BL/6 mice underwent bilateral renal ischemia-reperfusion injury (IRI), and kidneys were subjected to single-nucleus long-read RNA-seq. Sequencing libraries were prepared using the Single Cell 3' Reagent Kit (10x Genomics) and Ligation Sequencing Kit (Oxford Nanopore Technologies). Libraries were sequenced on PromethION 24 and basecalled with Dorado v0.9.0. Read mapping was performed using the EPI2ME workflow. R packages used in downstream analyses included Seurat and DTUrtle.
Results
A total of 1.3 billion reads were generated, with median read length of 765–884 bases across samples. Single-nucleus libraries yielded fewer reads assigned to isoforms but detected a greater number of isoform features compared to single-cell libraries, partly attributable to the abundance of mitochondrial gene-derived reads in single-cell libraries. In proximal tubule cells, genes associated with oxidative phosphorylation were downregulated after IRI not only at the gene expression level but also at the level of alternative splicing: protein-coding isoforms were downregulated whereas one or more non-protein-coding isoforms were upregulated in 21 genes. Beyond oxidative phosphorylation-associated genes, Fut9, encoding fucosyltransferase 9, exhibited a prominent isoform switch. Despite comparable total gene expression, the protein-coding isoform Fut9-201 was predominantly expressed in proximal tubule cells from healthy kidneys but markedly diminished following IRI. This finding was validated by quantitative PCR, which confirmed a 4-fold higher expression of Fut9-201 in healthy kidneys.
Conclusion
Single-nucleus long-read RNA-seq detected isoform-level regulation of gene expression in proximal tubule cells, providing deeper insight into transcriptomic regulation in response to acute injury that is invisible to conventional short-read approaches.