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 spatial long-read approaches often lack single-cell resolution and yield limited read lengths. Here, we introduce spatial ISOform sequencing (Spl-ISO-Seq), which reveals exons and polyadenylation sites with near-single-cell resolution. Spl-ISO-Seq selects long cDNAs and doubles to triples read lengths compared to standard preparations. Adding a highly specific software tool (Spl-ISOquant) and comparing human post-mortem pre-puberty (8-11 years) to post-puberty (16-19 years) visual cortex samples, we find that cortex harbors stronger splicing and poly(A)-site regulation than white matter. However, oligodendrocyte regulation is stronger in white matter. Among cortical layers, layer 4 has the most developmentally-regulated splicing changes in excitatory neurons and in poly(A) sites. We also find repeat elements downstream of developmentally-regulated layer 4 exons. Overall, alternative splicing changes are linked to post-synaptic structure and function. These results root developmental splicing changes during puberty in specific layers and cell types. More generally, our technologies enable exciting observations for any complex tissue.