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Psychrophilic proteases dramatically reduce single cell RNA-seq artifacts: A molecular atlas of kidney development
Author(s) -
Mike Adam,
Andrew Potter,
S. Steven Potter
Publication year - 2017
Publication title -
development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.15
H-Index - 36
eISSN - 1477-9129
pISSN - 0950-1991
DOI - 10.1242/dev.151142
Subject(s) - biology , proteases , gene expression , protease , psychrophile , cell , gene , rna , microbiology and biotechnology , single cell analysis , computational biology , enzyme , biochemistry
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.

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