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. Researchers from the Cincinnati Children’s Hospital Medical Center 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 they 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. They 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.
Venn diagram showing overlap of gene expression differences associated
with incubation at 37O C for varying time periods
This analysis focuses on five cell types, endothelial (Endo), podocytes (Pod), proximal tubule (Prox), loop of Henle (LOH), and cap mesenchyme progenitors. For each cell type the gene expression patterns of the cells incubated at 37oC were compared with the gene expression patterns of the same cell types generated using the cold active protease procedure (P < 0.001, FC > 2). Results are shown for 15, 30 and 60 min 37oC incubations. Cell type specific as well as shared gene expression changes are observed. The shared genes, showing change in more than one cell type, are thereby cross-validated. There are increasing numbers of these genes as a function of time of incubation at 37oC.