Psychrophilic proteases dramatically reduce single cell RNA-seq artifacts: A molecular atlas of kidney development
Open accessAbstract
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.
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Typical enzymatic digestion of mouse brain tissue induced a host of transcriptional alterations.
“Consistent with the reports from other tissues in mice, we found that typical enzymatic digestion of mouse brain tissue induced a host of transcriptional alterations34,43,44,65.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [65]
Emerging results from other tissues and cell types have begun to characterize dissociation artifacts following enzymatic digestion.
“However, emerging results from other tissues/cell types have begun to characterize dissociation artifacts following enzymatic digestion43–45,65,66.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [65]
The inhibitor-based protocol offers greater experimental and tissue-/cell-type flexibility than previously developed experimental and computational solutions.
“Our inhibitor-based protocol offers much greater experimental and tissue-/cell-type flexibility than previously developed experimental and computational solutions for this pervasive issue (see additional discussion of some alternative methods in Supplementary Note 7)33,34,45,65,67,68.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [65]
One drawback of an ice-cold dissociation is the decrease in efficiency of dissociation with certain tissues such as the brain.
“However, one drawback of an ice-cold dissociation is the decrease in efficiency of dissociation with certain tissues such as the brain (Adam et al., 2017;Denisenko et al., 2019;van den Brink et al., 2017).”
From Single-cell resolution analysis of the crosstalk between chemogenically activated astrocytes and microglia (2020) · cites this paper as Adam et al., 2017
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“However, one drawback of an ice-cold dissociation is the decrease in efficiency of dissociation with certain tissues such as the brain (Adam et al., 2017; Denisenko et al., 2019; van den Brink et al., 2017).”
Background
- Authors
- Adam, M., Potter, A. S., & Potter, S. S.
- Affiliations
- Cincinnati Children's Hospital Medical Center (US)
- Corresponding author
- Adam, Mike
- Venue
- Development
- Publisher
- The Company of Biologists
- Published
- 2017-01-01
- Volume
- 144
- Issue
- 19
- Pages
- 3625-3632
- ISSN
- 0950-1991 (ISSN-L)
- 1477-9129
- PMC
- PMC5665481
- OpenAlex
- W2752378502
- Cited by
- 343
- References
- 4
- Citations by year
- 2026: 14 · 2025: 33 · 2024: 44 · 2023: 46 · 2022: 44 · 2021: 55 · 2020: 41 · 2019: 41 · 2018: 23 · 2017: 2
- FWCI
- 11.96
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Development (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Funders
- National Institutes of Health (UO1DK107350)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- tissue dissociation, kidney development
- MeSH terms
- Animals, Kidney, Kidney Tubules, Proximal, Peptide Hydrolases, Temperature, Time Factors, Artifacts, Stromal Cells, Sequence Analysis, RNA, Gene Expression Regulation, Developmental, Gene Expression Profiling, Organogenesis, Mice, Single-Cell Analysis
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