Single-cell RNA-sequencing (scRNA-seq) has emerged in recent years as a breakthrough technology to understand RNA metabolism at cellular resolution. In addition to allowing new cell types and states to be identified, scRNA-seq can permit cell-type specific differential gene expression changes, pre-mRNA processing events, gene regulatory networks and single-cell developmental trajectories to be uncovered. More recently, a new wave of multi-omic adaptations and complementary spatial transcriptomics workflows have been developed that facilitate the collection of even more holistic information from individual cells. These developments have unprecedented potential to provide penetrating new insights into the basic neural cell dynamics and molecular mechanisms relevant to the nervous system in both health and disease.
University of Edinburgh researchers discuss this maturation of single-cell RNA-sequencing over the past decade, and review the different adaptations of the technology that can now be applied both at different scales and for different purposes. They conclude by highlighting how these methods have already led to many exciting discoveries across neuroscience that have furthered our cellular understanding of the neurological disease.
A recipe book for scRNA-seq
A) Cellular barcoding can be achieved by PCR or tagmentation-based late indexing of independent cDNA libraries prepared in separated chambers, or via early introduction of cell barcodes during the reverse transcription reaction. B) Amplification of low abundance cellular material can be achieved by in vitro transcription, PCR following a template-switching reverse transcription reaction, or rolling circle amplification. C) The choice of scRNA-seq workflow will determine the distinct coverage enrichment profiles observed along captured RNA transcripts. D) Independent cDNA libraries prepared in separated chambers can lead to full transcript coverage at reduced scale. Increased scaling can be achieved by increasing the number of ‘chambers’ from which libraries are prepared, and providing cellular indexes during reverse transcription such that early pooling of cellular transcriptomes can be achieved. The former can be achieved using massively parallel microwells, using microfluidic systems to rapidly generate thousands of oil droplet-based reaction chambers that encapsulate single cells, or employing the cells themselves as the reaction chambers for in situ library preparations. E) Starting material can be heterogeneous tissue or cell culture preparations. The characteristics of the input will determine whether whole cells or purified nuclei are to be profiled