The use of cold active proteases can dramatically reduce single cell RNA-seq gene expression artifacts

Single cell RNA-seq is a powerful methodology, but with important limitations. In particular, the process of enzymatic separation of cells at 37O C can be expected to result in artifact changes in gene expression patterns. We here describe a dissociation method that uses protease from a psychrophilic microorganism with high activity in the cold. The entire procedure is carried out at 6O C or colder, where mammalian transcriptional machinery is largely inactive. To test this method we carry out single cell RNA-seq on about 9,000 cells, comparing the results of the cold method with a method using 37O C incubations for multiple times. We show that the cold active protease method results in a great reduction in gene expression artifacts. Overall design: Whole mouse post natal day 1 kidney cells were dissassociated by either a cold active protease or an enzyme cocktail for varying lengths of time. The gene expression profiles of the four groups of cells were determined by drop-seq / RNA-seq.

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Adam M, Potter AS, Potter SS