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

• Type of Material: Article
• Authors: Simon A Hardwick, Weill Cornell Medicine; Wen Hu, Weill Cornell Medicine; Anoushka Joglekar, Weill Cornell Medicine; Li Fan, Weill Cornell Medicine; Paul G Collier, Weill Cornell Medicine; Careen Foord, Weill Cornell Medicine; Jennifer Balacco, The Rockefeller University; Samantha Lanjewar, Emory University; Maureen M Sampson, Emory University; Frank Koopmans, Vrije Universiteit Amsterdam; Audrey D Prjibelski, St. Petersburg State University; Alla Mikheenko, St. Petersburg State University; Natan Belchikov, Weill Cornell Medicine; Julien Jarroux, Weill Cornell Medicine; Anne Bergstrom Lucas, Agilent Technologies; Miklós Palkovits, Semmelweis University; Wenjie Luo, Weill Cornell Medicine; Teresa A Milner, Weill Cornell Medicine; Lishomwa C Ndhlovu, Weill Cornell Medicine; August B Smit, Vrije Universiteit Amsterdam; John Q Trojanowski, University of Pennsylvania; Virginia MY Lee, University of Pennsylvania; Olivier Fedrigo, Rockefeller University; Steven Sloan, Emory University; Dóra Tombácz, Szeged University; Elizabeth M Ross, Weill Cornell Medicine; Erich Jarvis, Rockefeller University; Zsolt Boldogkoi, Szeged University; Li Gan, Szeged University; Hagen U Tilgner, Weill Cornell Medicine
• Language: English
• Date: 2022-03-07
• Publisher: NATURE PORTFOLIO
• Publication Version: Final Published Version
• Copyright Statement: © The Author(s) 2022
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1038/s41587-022-01231-3
• Title of Journal or Parent Work: NATURE BIOTECHNOLOGY
• Volume: 40
• Issue: 7
• Start Page: 1082
• End Page: +
• Grant/Funding Information: Z.B. was supported by NKFIH K128247. D.T. was supported by FK128252.; J.Q.T. is supported by NIH grant U19 AG062418.; L.G. is supported by NIH grants R01AG072758, U54NS100717 and R01AG054214 and the JPB Foundation. T.A.M. is supported by NIH grants DA08259 and HL136520.; E.D.J. and O.F. are supported by funds from the Howard Hughes Medical Institute.; H.U.T. is supported by NIGMS grant 1R01GM135247-01, Brain Initiative grant 1RF1MH121267-01, NIDA grant U01 DA053625-01 and the Feil Family Foundation. M.E.R. is supported by NIH grants 1R01NS105477, P01HD067244 and U54NS117170 and the Feil Family Foundation.; Computational analysis was performed with the help of the Research Park of St. Petersburg State University Computing Center.; S.A.H. is supported by an Australian NHMRC Early Career Fellowship (APP1156531).; L.C.N. is supported, in part, by the NIMH, NIDA, NINDS, NIDDK, NHLBI and NIAID under award number UM1AI164599 and by the NIDA under award number U01 DA53625 (to L.C.N., H.U.T. and T.A.M.).; A.M. and A.D.P. are supported by St. Petersburg State University (grant ID PURE 73023672).; M.P. is supported by the Hungarian Brain Research Program (2017-1.2.1-NKP-2017-00002, NAP2.0) through the Human Brain Tissue Bank at Semmelweis University.; S.A.S. is supported by NIMH grant R01MH125956 and the Brain and Behavior Foundation (grant 28172).
• Keywords: ASSOCIATION; TRANSCRIPTOMIC CELL-TYPES; Biotechnology & Applied Microbiology; DIVERSITY; CORTEX; MICROEXONS; Science & Technology; Life Sciences & Biomedicine; SEQ
• Research Categories: Health Sciences, Medicine and Surgery

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.