Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development

• Published in: Development (Cambridge) Vol. 144; no. 19; pp. 3625 - 3632
• Main Authors: Adam, Mike, Potter, Andrew S, Potter, S Steven
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Ltd 01.10.2017
• Subjects: Animals; Freezing; Gene expression; Gene Expression Profiling; Gene Expression Regulation, Developmental; Kidney - embryology; Kidney - metabolism; Kidney Tubules, Proximal - cytology; Kidney Tubules, Proximal - embryology; Kidneys; Mice; Nephrons; Organogenesis; Peptide Hydrolases - metabolism; Proteinase; Ribonucleic acid; RNA; Rodents; Sequence Analysis, RNA - methods; Single-Cell Analysis - methods; Stromal Cells - cytology; Stromal Cells - metabolism; Techniques and Resources; Temperature; Time Factors; Transcription; Single cell; Artifacts; Cell dissociation; RNA-seq; Kidney development
• ISSN: 0950-1991; 1477-9129
• DOI: 10.1242/dev.151142

Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively 'freezing in' the gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.