A spatial long-read approach at near-single-cell resolution reveals developmental regulation of splicing and polyadenylation sites in distinct cortical layers and cell types.

Genome-wide single-cell and spatial long-read approaches have gained traction, but mostly lack single-cell resolution - and yield reduced read lengths. Here, we introduce spatial ISOform sequencing (Spl-ISO-Seq), which reveals exons and polyadenylation (polyA) sites from long reads with near-single-cell resolution. Spl-ISO-Seq selects for long cDNAs doubling to tripling read lengths. Adding a highly specific software tool (Spl-ISOquant) and comparing pre-puberty samples of the visual cortex (8-11 years) to post-puberty samples (16-19 years), we find that cortical layers harbor stronger splicing and poly(A)-site regulation than the adjacent white matter, with enrichment of multiple protein-domain types. For oligodendrocytes however, developmental splicing changes are stronger in white matter. Among cortical layers, layer 4 has the strongest changes in alternative-exon inclusion, an observation caused by excitatory neurons, and poly(A) sites. Overall, alternative splicing changes are linked to synapses - specifically at the post-synapse. Age-linked splicing changes in layers 1-3 and 4 are associated with autism spectrum disorder but not with schizophrenia, amyotrophic lateral sclerosis and Alzheimer's diseases. These results root developmental splicing changes during puberty and the resulting protein changes in specific layers and cell types. More generally, our new technologies enable new observations for any complex tissue.