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.
Highlights
Concurrent with the development of single-cell RNA sequencing[1–3], long-read approaches enabled complete isoform analysis[4–8]
SnISOr-Seq can detect multiple splicing events in barcoded long reads, which might originate from genuine polyA sites as well as internal polyA-rich regions
Using SnISOr-Seq, we investigate how distinct transcript elements—alternative transcription start sites (TSSs), exons and polyA sites—are combined into full-length isoforms in the human brain and determine the cell-type-specific basis of coordination events
Summary
Concurrent with the development of single-cell RNA sequencing[1–3], long-read approaches enabled complete isoform analysis[4–8]. Single-nuclei datasets contain many partially or fully unspliced RNAs, leading to many reads derived from purely intronic regions These reads are reverse-transcribed from genomically encoded polyadenylation (polyA)-rich regions or through artifacts and are usable for gene count and ‘RNA velocity’ analyses[16–18]. Such intronic reads cannot inform on complete isoforms Another problem for long-read sequencing of 10x Genomics single-nuclei and single-cell libraries are molecules lacking polyA tails, barcodes and Illumina adaptors (Fig. 1b). We employ linear/asymmetric PCR, amplifying full-length cDNAs from the 10x Genomics partial-read[1], near which polyA tails and barcodes reside This step enriches for polyA-tail-containing and barcode-containing molecules (Fig. 1c). Using SnISOr-Seq, we investigate how distinct transcript elements—alternative transcription start sites (TSSs), exons and polyA sites—are combined into full-length isoforms in the human brain and determine the cell-type-specific basis of coordination events. Autism spectrum disorder (ASD)-associated exons show markedly different behavior than schizophrenia-associated and amyotrophic lateral sclerosis (ALS)-associated exons, with the caveat that distinct methods defined the exons associated to each disease
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