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

Development. 2017 Oct 1;144(19):3625-3632. doi: 10.1242/dev.151142. Epub 2017 Aug 29.


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 in vivo 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.

Keywords: Artifacts; Cell dissociation; Kidney development; RNA-seq; Single cell.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Artifacts*
  • Gene Expression Profiling
  • Gene Expression Regulation, Developmental
  • Kidney / embryology*
  • Kidney / metabolism
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / embryology
  • Mice
  • Organogenesis*
  • Peptide Hydrolases / metabolism*
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*
  • Stromal Cells / cytology
  • Stromal Cells / metabolism
  • Temperature
  • Time Factors


  • Peptide Hydrolases