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

• DOI: 10.1038/s41587-022-01231-3
• Publication Date: 2022-03-07T17:03:48Z
• Authors: Simon A. Hardwick; Wen Hu; Anoushka Joglekar; Li Fan; Paul G. Collier; Careen Foord; Jennifer Balacco; Samantha Lanjewar; Maureen McGuirk S...; Frank Koopmans; Andrey D. Prjibelski; Alla Mikheenko; Natan Belchikov; Julien Jarroux; Anne Bergstrom Lucas; Miklós Palkovits; Wenjie Luo; Teresa A. Milner; Lishomwa C. Ndhlovu; August B. Smit; John Q. Trojanowski; Virginia M. Y. Lee; Olivier Fedrigo; Steven A. Sloan; Dóra Tombácz; M. Elizabeth Ross; Erich Jarvis; Zsolt Boldogkői; Li Gan; Hagen U. Tilgner
• Keywords: Alternative Splicing; Sequence Analysis, RNA; Brain; Humans; Protein Isoforms; RNA; 01.06. Biológiai tudományok; 03.01. Általános orvostudomány; SDG 10 - Reduced Inequalities; Exons; Article

AbstractSingle-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.