Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue

Nat Biotechnol. 2022 Jul;40(7):1082-1092. doi: 10.1038/s41587-022-01231-3. Epub 2022 Mar 7.

Abstract

Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Alternative Splicing / genetics
  • Brain* / metabolism
  • Exons / genetics
  • Humans
  • Protein Isoforms / genetics
  • RNA* / genetics
  • Sequence Analysis, RNA

Substances

  • Protein Isoforms
  • RNA