Many neurological diseases like schizophrenia stem partly from aberrant early brain development. Gene regulation is notably complex in the brain, and processes like alternative splicing mean many RNA isoforms exist and often contribute differently to neurodevelopment. The optimal method to capture different RNA isoforms involves sequencing long-reads. However, landmark studies of human neurodevelopment have analysed short-reads. Despite detecting gene expression, these studies were unable to capture isoform-level data. This is what we seek to address. We plan to sequence long-read cDNA (generated from RNA) at the single-cell level in the post-mortem prefrontal cortex (PFC), across an unprecedented range of neurodevelopmental timepoints. Our project will elucidate how the transcriptome shapes crucial brain development.
As an initial pilot study, we mapped the RNA isoform landscape of 9,988 cells from the PFC of a 2-day-old post-mortem healthy female. After single-nuclei capture and preparation (10x Chromium Next GEM Single Cell 3’ kit), we performed Oxford Nanopore (ONT LSK-114) long-read cDNA sequencing and analysed the data using Seurat and our custom bioinformatics pipeline FLAMES. Using these tools, we assessed isoform expression and diversity across cell types.
We identified 19 major cell types, with three broad groups including excitatory neurons, GABAergic interneurons and non-neuronal cells. We detected 38,825 expressed genes and 141,111 isoforms, including 156 novel isoforms. 38% of isoforms were differentially expressed between cell types, with excitatory neurons showing the greatest diversity. We also found neuropsychiatric risk genes typically expressed many mRNA isoforms, including ANK2 and KMT2C.
This is the first single-cell study of RNA isoforms in the post-natal developing human brain. We show extensive isoform diversity, particularly in neuropsychiatric risk genes, and highlight isoforms of interest. Expanding this study across developmental stages will reveal how RNA diversity may contribute to brain health and disease.