SnISOr-Seq – single-nuclei isoform RNA sequencing

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

Fig. 1

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).

Hardwick SA, Hu W, Joglekar A, Fan L, Collier PG, Foord C, Balacco J, Lanjewar S, Sampson MM, Koopmans F, Prjibelski AD, Mikheenko A, Belchikov N, Jarroux J, Lucas AB, Palkovits M, Luo W, Milner TA, Ndhlovu LC, Smit AB, Trojanowski JQ, Lee VMY, Fedrigo O, Sloan SA, Tombácz D, Ross ME, Jarvis E, Boldogkői Z, Gan L, Tilgner HU. (2022) Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue. Nat Biotechnol [Epub ahead of print]. [article]

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