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. A team led by researchers at Weill Cornell Medicine have developed 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. The researchers applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. They 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, the researchers 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.
Overview of the SnISOr-Seq approach
a, Barcoded cDNA library of nuclei isolated from frozen human brain tissue. b, Three main types of molecules generated: spliced barcoded (known and novel isoforms), unspliced barcoded (exclusively intronic nucleotides) and incomplete cDNA without a cellular barcode. c, Linear/asymmetric PCR (‘LAP’) is used to selectively amplify barcoded cDNA. d, Probe-based exome capture (‘CAP’) step is applied to filter out purely intronic cDNA molecules. e, Molecules are sequenced on a long-read sequencer (PacBio and ONT).