biostudies-literatureHardwick SAHungarian Brain Research Program, The Human Brain Tissue Bank, Semmelweis UniversityU.S. Department of Health & Human Services | NIH | National Institute of General Medical SciencesNICHD NIH HHSSemmelweis EgyetemNIA NIH HHSHoward Hughes Medical InstituteNIMH NIH HHSNHLBI NIH HHSJPB FoundationDepartment of Health | National Health and Medical Research CouncilU.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNINDS NIH HHSSaint Petersburg State UniversityNemzeti Kutatási, Fejlesztési és Innovációs HivatalNIDA NIH HHSNIAID NIH HHSU.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious DiseasesU.S. Department of Health & Human Services | NIH | National Institute on Drug AbuseBrain and Behavior Research FoundationU.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood InstituteU.S. Department of Health & Human Services | NIH | National Institute of Mental HealthFeil Family FoundationU.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and StrokeU.S. Department of Health & Human Services | National Institutes of HealthNIGMS NIH HHS1082-1092https://www.ebi.ac.uk/biostudies/studies/S-EPMC9287170biostudies-literatureUnknown40(7)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 exoNature biotechnologySingle-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue.PMC9287170FK128252K128247R01MH125956R01 AG051390APP1156531R01 GM135247R01 HL136520T32 DA0390801R01GM135247-01PURE 73023672R01AG054214R01 NS105477HL136520U01 DA053625-01U01 DA0536251R01NS105477R01 DA008259U54 NS10071728172RF1 MH121267P01HD067244DA08259R01AG051390U54NS100717UM1AI164599R01 NS123562R01 AG054214R01 AG072758U19 AG062418P01 HD067244R56 AI164599U54NS11717R01 MH125956U54 NS117170U01 DA53625UM1 AI1645591RF1MH121267-012017-1.2.1-NKP-2017-00002Prjibelski ADLanjewar SHu WTilgner HUMilner TATrojanowski JQJoglekar APalkovits MFedrigo OTombacz DFoord CNdhlovu LCFan LBalacco JSampson MMLee VMYSmit ABSloan SAHardwick SAJarvis ERoss MEBelchikov NKoopmans FBoldogkoi ZGan LMikheenko ACollier PGLucas ABLuo WJarroux JfalseSingle-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue.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 exo2022-01-01T00:00:00Z2022 Jul2026-05-09T21:54:25.399Z2025-02-19T02:33:08.603ZS-EPMC92871703525681510.1038/s41587-022-01231-3