Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq) allow transcriptomic profiling of thousands of cells from a renal biopsy specimen at a single-cell resolution. Both methods are promising tools to unravel the underlying pathophysiology of glomerular diseases. KU Leuven researchers provide 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 biopsy specimens 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 before single-cell analysis is challenging because no effective standardized pipelines are available. Fourth, the current warm cell-dissociation protocols may damage glomerular cells and induce transcriptional artifacts, which can be minimized by using cold dissociation techniques at the cost of less efficient cell dissociation. Finally, snRNA-seq methods may be superior to scRNA-seq in isolating glomerular cells; however, the efficacy of snRNA-seq on core needle biopsy specimens remains to be proven. The field of single-cell omics is rapidly evolving, and the integration of these techniques in multiomics assays will undoubtedly create new insights in the complex pathophysiology of glomerular diseases.
General workflow of scRNA-seq and snRNA-seq experiments
Renal tissue is mechanically and enzymatically dissociated into a single-cell or single-nucleus suspension. In scRNA-seq, viable single cells are dissociated from their extracellular matrix; in snRNA-seq, stronger dissociation techniques are used to dissociate nuclei from the cells.3 Next, cells or nuclei are sorted into a microtiter plate or loaded into a microfluidics device containing integrated fluidic circuits (IFCs), nanowells, or droplets, ready for subsequent processing steps.
Individual cells or nuclei are lysed and transcripts from single cells or single nuclei are captured using poly[T] oligonucleotide primers that hybridize with the polyadenylated (poly[A]) mRNA. These primers may also contain other oligonucleotide sequences, including a cell barcode (BC), unique molecular identifier (UMI), and adaptor sequence for PCR or RNA polymerase promotor (T7 promotor) for in vitro transcription (IVT).3 Next, a reverse transcription (RT) reaction synthesizes the first and second strand of cDNA, which is subsequently amplified by either PCR or IVT. Amplified cDNA molecules are fragmented (using enzymatic tagmentation or chemical fragmentation) and ligated with adaptors for sequencing. A sequence library is created and, finally, using advanced bioinformatics analysis, sequencing data are translated into cellular expression patterns, leading to the identification of distinct cell clusters.