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

Abstract

Single cell RNA-seq is a powerful methodology. 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 artifact changes in gene expression patterns. We here 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, where mammalian transcriptional machinery is largely inactive, thereby effectively “freezing in” the in vivo gene expression patterns. To test this method we carried out RNA-seq on 20,424 single cells from P1 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.