Transcriptome analysis enables the study of gene expression in human tissues and is a valuable tool to characterise liver function and gene expression dynamics during liver disease, as well as to identify prognostic markers or signatures, and to facilitate discovery of new therapeutic targets. In contrast to whole tissue RNA sequencing analysis, single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics enables the study of transcriptional activity at the single cell or spatial level. ScRNA-seq has paved the way for the discovery of previously unknown cell types and subtypes in normal and diseased liver, facilitating the study of rare cells (such as liver progenitor cells) and the functional roles of non-parenchymal cells in chronic liver disease and cancer. By adding spatial information to scRNA-seq data, spatial transcriptomics has transformed our understanding of tissue functional organisation and cell-to-cell interactions in situ. These approaches have recently been applied to investigate liver regeneration, organisation and function of hepatocytes and non-parenchymal cells, and to profile the single cell landscape of chronic liver diseases and cancer. Researchers from the Université de Strasbourg discuss the principles and technologies behind scRNA-seq and spatial transcriptomic approaches, highlighting the recent discoveries and novel insights these methodologies have yielded in both liver physiology and disease biology.
Main steps in scRNA-seq workflows and comparison of the most widely used protocols
Smart-seq2 and CEL-Seq2 are performed in 96 or 384-well plates after FACS sorting, while droplet systems (e.g. 10X Chromium and Drop-Seq) couple cells with barcoded beads containing UMI and primers forming water-in-oil droplets via a continuous oil flow. Reverse transcription and cDNA amplification are performed by PCR in Smart-Seq and 10X Chromium and by IVT in CEL-Seq2. In CEL-Seq2 and 10X Chromium protocols, UMI and cell-specific barcodes are added during reverse transcription to allow the pooling of the subsequent steps. Libraries are prepared by fragmentation in CEL-Seq2 and by tagmentation with or without 3′enrichment in Smart-Seq2 and 10X Chromium. Gene coverage is full-length in Smart-seq2 whereas in CEL-Seq2 and 10X Chromium only the 3′ part of the gene is sequenced. IVT, in vitro transcription; scRNA-seq, single-cell RNA-sequencing; UMI, unique molecule identifier.