Long-Read Single-Cell RNA Sequencing Reveals the Alternative Splicing Landscape across Mouse Kidney Epithelial Cells

Background: There is surprisingly little known regarding alternative splicing (AS) in kidney health and disease, and to date no study has reported cell-specific AS patterns using long-read single-cell RNA-seq (scRNA-seq) in the kidney. Long-read sequencing more accurately identifies full-length gene isoforms to better determine AS that differentiates kidney cell types and can identify cell type-specific transcripts to refine kidney cell identities. Methods: A 15-week-old C57BL/6J male mouse kidney was dissociated into single cells and barcoded using the 10X Genomics Single Cell 3’ GEM kit (v3.1). The library was prepared using Oxford Nanopore Technologies (ONT) PCR-cDNA SQK-PCS111 kit and sequenced on a PromethION flow cell. QC and gene/transcript quantification was performed using ONT’s EPI2ME Labs wf-single-cell workflow. Seurat was used for gene- and transcript-level PCA and clustering. DTUrtle was used to determine differential transcript usage (DTU) between cell types. Results: Gene-level clustering identified known kidney cell types and was used to determine differentially expressed transcripts (DET) and DTU. Long-read scRNA-seq detected differential transcript usage (DTU) in all kidney cell types, with most DTU events observed between epithelial cells such as the proximal tubule (PT), thick ascending limb (TAL), and distal convoluted tubule (DCT). We compared all PT cells to all other cell types in the dataset and found isoform Miox-201 (myo-inositol oxygenase) as the most significant DET and expression was highly specific to PT cells compared to all other cell types, identifying a novel PT cell-specific isoform. DTU analysis between PT-S1 and TAL identified 37 genes and 76 transcripts with significant DTU. DTU genes between these two cell types were in pathways related to Na/K transport, cellular communication, and cell adhesion. The isoform Cldn10-201 (claudin-10) had a 76% increase in usage in PT-S1 versus TAL. This isoform switch results in preferring anions over cations, which corresponds to its function along the nephron. Conclusion: We show for the first time the single-cell full-length isoform landscape of the murine kidney. Cell type-dependent DTU adds an additional layer of gene regulation, therefore, understanding the mechanisms of AS in the kidney could lead to new targets for future therapeutics to treat kidney diseases. Funding: NIDDK Support