Almost all human genes express multiple mRNA products (RNA isoforms), which can have different or even opposing functions. The expression of specific RNA isoforms is central to many aspects of brain development and disease. While RNA isoforms and their expression profiles are particularly diverse in brain, their cell-type specificity and individual functions are poorly understood.
To address this, we developed methods and software for long-read single-cell RNA-seq (LR scRNA-seq), enabling the profiling of known and novel RNA isoforms at the single cell level, elucidating how isoform expression varies both within and between cell types. Here we introduce a faster and more accurate version of our widely used FLAMES LR scRNA-seq analysis package and apply it to study neurogenesis in 2D neuronal cultures and differentiation of cortical organoids. LR scRNA-seq achieved high resolution identification of cell types, including subtypes of radial glial progenitors and excitatory neurons. We identified >170,000 unique isoforms, including >10,000 which were previously unknown. We discovered thousands of differentially expressed isoforms associated with synaptic transmission, neuronal projection, axonogenesis, and neuron development. This included genes such as PKM and GPM6A, which showed ubiquitous gene expression but cell-type-specific isoform expression. Our work demonstrates the power of LR scRNA-seq for isoform-level characterization. Uncovering the expression and function of isoforms will be essential for our molecular understanding of brain development and disease pathogenesis.