Single-cell brain RNA isoforms across anatomical structures, postnatal development and brain diseases – or – isoforms in time and space

Complex tissue includes diverse cell types employing distinct RNA isoforms. To untangle full-length cell-type specific brain isoforms, we developed single-cell long-read technology for many thousands of cells (from previous approaches for 10-100 cells) in fresh tissues (ScISOr-Seq; Gupta, Collier et al, 2018¹) and in frozen tissues (SnISOr-Seq; Hardwick, Hu, Joglekar et al, 2022²). These approaches revealed the rules of combination of TSSs, alternative exons and poly(A) sites and their cell-type specificity. Autism-associated exons (as previously described) but also FTD-associated exons are highly variably-used across cell types². For spatial resolution, we developed spatially-barcoded isoform sequencing with 60um (Joglekar et al, 2021³), 10um (Foord, Prjibelski, Hu et al 2025⁴) and 220nm (Michielsen, Prjibelski, Foord et al, biorxiv5) spots, showing that often isoform switches correlate with precise boundaries of brain structures (e.g., choroid plexus to hippocampus). However, genes including Snap25, use a gradient of exon inclusion through the brain³. Choroid plexus epithelial cells show a dramatically distinct isoform profile, which originates most strongly from TSS usage3. During human puberty, layer4-excitatory splicing is more regulated than in other cortical layers – probably influenced by retroviral sequences^4. More generally, we can now detect isoform-expression variability that does not correspond to known brain structures⁵.

For the NIH Brain Initiative, we have mapped single-cell isoforms across development, brain regions and species. Neurotransmitter release and reuptake as well as synapse turnover genes harbor variability in the same cell type across anatomical regions but the same cell type traced across development shows more isoform variability than across adult anatomical regions. Moreover, most cell-type-specific exons in adult mouse hippocampus behave similarly in human hippocampi. However, human brains have evolved additional cell-type specificity in splicing (Joglekar et al, 2024⁶). Additionally, the concurrent measurement of chromatin and splicing patterns in post-mortem human brain shows broadly-speaking convergent dysregulation of both modalities in similar cell types in Alzheimer’s disease but more divergence between both modalities in evolution (Hu, Foord, Hsu et al, 2025⁷). Finally, we have advanced our understanding of error sources of PacBio and ONT (Mikheenko, Prjibelski et al, 2022⁸) and implemented highly accurate long-read software (Prjibelski, Mikheenko et al, 2023⁹).

¹ Gupta et al, Nat Biotechnol, 2018
² Hardwick et al, Nat Biotechnol, 2022
³ Joglekar et al, Nat Commun, 2021
⁴ Foord et al, Nat Commun, 2025
⁵ Michielsen et al, biorxiv, 2025
⁶Joglekar et al, Nat Neurosci, 2024
⁷ Hu et al, Nat Biotechnol, 2025
⁸ Mikheenko et al, Genome Res, 2022
⁹ Prjibelski et al, Nat Biotechnol, 2023