ScISOr-Seq – single-cell isoform RNA sequencing

Full-length isoform sequencing has advanced our knowledge of isoform biology. However, apart from applying full-length isoform sequencing to very few single cells, isoform sequencing has been limited to bulk tissue, cell lines, or sorted cells. Single splicing events have been described for <=200 single cells with great statistical success, but these methods do not describe full-length mRNAs. Single cell short-read 3′ sequencing has allowed identification of many cell sub-types, but full-length isoforms for these cell types have not been profiled. Using a new method of single-cell-isoform-RNA-sequencing (ScISOr-Seq) researchers from Weill Cornell Medicine determine isoform-expression in thousands of individual cells from a heterogeneous bulk tissue (cerebellum), without specific antibody-fluorescence activated cell sorting. They elucidate isoform usage in high-level cell types such as neurons, astrocytes and microglia and finer sub-types, such as Purkinje cells and Granule cells, including the combination patterns of distant splice sites, which for individual molecules requires long reads. The researchers produce an enhanced genome annotation revealing cell-type specific expression of known and 16,872 novel (with respect to mouse Gencode version 10) isoforms (see isoformatlas.com).

(A) Outline of the ScISOr-Seq approach. (B) TSNE-plot depicting cell clusters, marker genes and names given to clusters, including: Bergman glia (BG), External  granule  cell  layer  neurons  (EGL),  Internal  granule  cell  layer  and  other  neurons in the interior of the cerebellum (IGL), two clusters of Purkinje cell layer neurons   (PCL),   oligodendrocyte   progenitor   cells   (OPCs),   Atoh1+   neuronal   progenitors, Ptf1a+ neuronal progenitors and other neuronal progenitors (NPCs)(C) In-situ hybridization images from the Allen Brain Atlas depicting expression of marker genes in specific layers. (D) Expression patterns of selected marker genes across cell types.