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PreprintCited by 686Single-cell and spatial transcriptomicsGenomics and Phylogenetic StudiesMolecular Biology Techniques and Applications

Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows

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Denisenko, Elena, Guo, Belinda B., Jones, Matthew, Hou, Rui, de Kock, Leanne, Lassmann, Timo, Poppe, Daniel, Clement, Olivier +3 more·2019·Genome biology
DOI 10.1101/832444PMC PMC7265231

Preprint — published as 10.1186/s13059-020-02048-6 · Paper page

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Abstract

Single-cell and single-nucleus RNA sequencing have been widely adopted in studies of heterogeneous tissues to estimate their cellular composition and obtain transcriptional profiles of individual cells. However, the current fragmentary understanding of artefacts introduced by sample preparation protocols impedes the selection of optimal workflows and compromises data interpretation. To bridge this gap, we compared performance of several workflows applied to adult mouse kidneys. Our study encompasses two tissue dissociation protocols, two cell preservation methods, bulk tissue RNA sequencing, single-cell and three single-nucleus RNA sequencing workflows for the 10x Genomics Chromium platform. These experiments enable a systematic comparison of recovered cell types and their transcriptional profiles across the workflows and highlight protocol-specific biases important for the experimental design and data interpretation.

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  • 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 Denisenko et al., 2019

    The Discussion (page not provided) describes lower efficiency in releasing hard-to-dissociate cell types as a possible drawback of cold dissociation, using kidney loop-of-Henle cells as a tentative example. It does not establish reduced dissociation efficiency in brain tissue; the study used mouse kidneys. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

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Authors
Denisenko, E., Guo, B. B., Jones, M., Hou, R., de Kock, L., Lassmann, T., Poppe, D., Clement, O., Simmons, R. K., Lister, R., & Forrest, A. R. R.
Affiliations
  • The University of Western Australia (AU)
  • The Kids Research Institute Australia (AU)
Corresponding author
Forrest, Alistair R. R.
ORCID
Venue
Genome biology
Publisher
openRxiv
Published
2019-11-06
Volume
21
Issue
1
Pages
130-130
ISSN
1465-6906 (ISSN-L)
DOI
10.1101/832444
PMC
PMC7265231
OpenAlex
W3030739213
Cited by
686
References
0
Citations by year
2026: 138 · 2025: 96 · 2024: 118 · 2023: 118 · 2022: 100 · 2021: 83 · 2020: 33
FWCI
27.50
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Genome biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • UWA Profiles and Research Repository (UWA) (submittedVersion, cc-by)
  • Adelaide Research & Scholarship (AR&S) (University of Adelaide) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Conquer Cancer Foundation
  • Australian Government
  • Australian Cancer Research Foundation
  • Government of Western Australia
  • Medical Research Council
  • National Health and Medical Research Council (1154524)
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, single-cell RNA sequencing, single-nucleus RNA sequencing, cryopreservation, methanol fixation, kidney, cell type composition
MeSH terms
Animals, Cell Separation, Kidney, Male, Mice, Inbred C57BL, Stress, Physiological, Cryopreservation, Sequence Analysis, RNA, Single-Cell Analysis

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