Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue
Simon A. Hardwick, Wen Hu, Anoushka Joglekar, Li Fan, Paul Collier, Careen Foord, Jennifer Balacco, Samantha N. Lanjewar, Maureen M. Sampson, 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 Fédrigo, Steven A. Sloan, Dóra Tombácz, M. Elizabeth Ross, Erich D. Jarvis, Zsolt Boldogkői, Li Gan, Hagen Tilgner
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.