Single-cell splice isoform usage reveals distinct axes of cellular identity and senescence

Alternative splicing greatly expands the diversity of gene products encoded by the human genome. Single-cell transcriptomic atlases have characterized human cell types through gene-level expression, but short-read sequencing has limited the ability to resolve full-length isoforms and their functional consequences. Here, we present a cross-tissue single-cell long-read isoform atlas spanning 26 human tissues. We identify hundreds of thousands of novel isoforms along with their cell-type-specific usage, and discover that over one-third of expressed isoforms are absent from existing reference databases. We further demonstrate that isoform usage is a structured, measurable axis of cellular identity that is distinct from gene expression. Applying this framework to cellular senescence, we resolve p16 ^INK4a and p14 ^ARF transcripts from the CDKN2A locus in individual cells and uncover cell-type-dependent isoform remodeling associated with the p16 ^INK4a senescence program. This isoform-resolved single-cell atlas offers a versatile framework to dissect the cellular logic of isoform regulation in senescence and beyond.