Single-cell RNA cap and tail sequencing (scRCAT-seq) reveals subtype-specific isoforms differing in transcript demarcation

The differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Gene isoforms allow a single gene diverse functions across different cell types, and isoform dynamics allow different functions over time. However, methods to efficiently identify and quantify RNA isoforms genome-wide in single cells are still lacking. Here, we introduce single cell RNA Cap And Tail sequencing (scRCAT-seq), a method to demarcate the boundaries of isoforms based on short-read sequencing, with higher efficiency and lower cost than existing long-read sequencing methods. In conjunction with machine learning algorithms, scRCAT-seq demarcates RNA transcripts with unprecedented accuracy. We identified hundreds of previously uncharacterized transcripts and thousands of alternative transcripts for known genes, revealed cell-type specific isoforms for various cell types across different species, and generated a cell atlas of isoform dynamics during the development of retinal cones. Most single-cell RNA sequencing methods have limited ability to profile the transcriptome at isoform resolution. Here the authors develop scRCAT-seq to characterize the 5′- and 3′-ends of transcripts in single cells, and identify cell-type specific alternative transcript isoforms.