Current Methodological Challenges of Single-Cell and Single-Nucleus RNA-Sequencing in Glomerular Diseases.

Abstract

Single-cell RNA-sequencing (scRNA-seq) and single-nucleus RNA-sequencing (snRNA-seq) allow transcriptomic profiling of thousands of cells from a renal biopsy at single-cell resolution. Both methods are promising tools to unravel the underlying pathophysiology of glomerular diseases. This review provides an overview of the technical challenges that should be addressed when designing single-cell transcriptomics experiments that focus on glomerulopathies. The isolation of glomerular cells from core needle biopsies for single-cell transcriptomics remains difficult and depends upon five major factors. First, core needle biopsies generate little tissue material and several samples are required to identify glomerular cells. Second, both fresh and frozen tissue samples may yield glomerular cells, although every experimental pipeline has different (dis)advantages. Third, enrichment for glomerular cells in human tissue prior to single-cell analysis is challenging as no effective standardized pipelines are available. Fourth, the current warm cell dissociation protocols may damage glomerular cells and induce transcriptional artefacts, which can be minimized by using cold dissociation techniques at a cost of less efficient cell dissociation. Finally, snRNA-seq methods may be superior to scRNA-seq in isolating glomerular cells, although its efficacy on core needle biopsies remains to be proven. The field of single-cell omics is rapidly evolving and the integration of these techniques in multi-omics assays will undoubtedly create new insights in the complex pathophysiology of glomerular diseases.