Multi-site Assessment of Methods for Cell Preservation Upstream of Single Cell RNA Sequencing Article Swipe
Fred Kolling
,
Jessica W. Podnar
,
Owen M. Wilkins
,
Christophe Fraser
,
Madolyn L. MacDonald
,
Shawn W. Polson
,
Zachary T. Herbert
,
Sridar V. Chittur
,
Andrew Hayden
,
Marcy Kuentzel
,
Michael Heinz
,
Gabriella Huerta
,
Holly Stevenson
,
Aditi Karmakar
,
Catrina Fronick
,
Lisa Cook
,
Sean Vargas
,
Xiaoling Xuei
,
Patrick McGuire
,
Molly J. Zeller
,
Yanping Zhang
,
Ru Dai
,
Xinkun Wang
,
Ching Man Wai
,
Jyothi Thimmapuram
,
Devender Arora
,
Tania Mesa
,
Jun Fan
,
Yuriy O. Alekseyev
,
Felice Cervone
,
Christopher Williams
,
Nickolas G. Gorham
,
Alexander Lemenze
,
Sara Goodwin
,
Jonathan Preall
,
Charles A. Whittaker
·
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1101/2025.10.24.684427
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1101/2025.10.24.684427
Single cell RNA sequencing (scRNA-seq) is a revolutionary technique to identify cell types and their molecular phenotype in heterogeneous biological specimens. ScRNA-seq typically requires fresh, high quality single cell suspensions that are processed immediately to preserve their molecular profiles. This presents a challenge for samples with long preparation times and prevents collection at remote sites lacking the required instrumentation for sample processing. Recently, several commercial assays have been released that enable sample preservation at the time of collection either via fixation or cryopreservation, allowing for sample processing to occur months after the initial collection. The Association of Biomolecular Research Facilities’ (ABRF) DNA Sequencing (DSRG) and Genomics Bioinformatics (GBiRG) Research Groups have undertaken a cross-platform, multi-site study to assess the performance and reproducibility of three platforms: a) 10x Genomics FLEX, b) Parse Bioscience Evercode WT v2 and c) Honeycomb Bio HIVE. Total leukocytes were isolated from a single healthy individual using the EasySep RBC depletion reagent. Cells were then characterized by collecting a 21-color flow cytometry dataset for reference and the remaining material was used for scRNA-seq procedures where different sites then processed either the fixed or cryopreserved cells for each method. We evaluated performance of each method across traditional scRNA-seq quality control metrics and analysis applications, including gene/transcript detection sensitivity, cell type discovery and annotation, and differential expression. We demonstrate that data from the methods tested can be effectively integrated and produce concordant results with regard to cell type annotation and relative abundance, though we observe platform-specific differences in the expression of a subset of genes. Preservation-based methods also show better retention of fragile granulocyte populations compared with fresh samples processed using the 10x 3’ workflow. The improvements to preservation methods are changing the way research is conducted and our thorough investigation into the performance of each method provides a valuable resource to help scientists determine the most appropriate single cell preservation workflow given their sample collection logistics and laboratory infrastructure constraints.
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Metadata
- Type
- preprint
- Language
- en
- Landing Page
- https://doi.org/10.1101/2025.10.24.684427
- OA Status
- green
- References
- 38
- OpenAlex ID
- https://openalex.org/W4415536007
All OpenAlex metadata
Raw OpenAlex JSON
- OpenAlex ID
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https://openalex.org/W4415536007Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1101/2025.10.24.684427Digital Object Identifier
- Title
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Multi-site Assessment of Methods for Cell Preservation Upstream of Single Cell RNA SequencingWork title
- Type
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preprintOpenAlex work type
- Language
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enPrimary language
- Publication year
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2025Year of publication
- Publication date
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2025-10-24Full publication date if available
- Authors
-
Fred Kolling, Jessica W. Podnar, Owen M. Wilkins, Christophe Fraser, Madolyn L. MacDonald, Shawn W. Polson, Zachary T. Herbert, Sridar V. Chittur, Andrew Hayden, Marcy Kuentzel, Michael Heinz, Gabriella Huerta, Holly Stevenson, Aditi Karmakar, Catrina Fronick, Lisa Cook, Sean Vargas, Xiaoling Xuei, Patrick McGuire, Molly J. Zeller, Yanping Zhang, Ru Dai, Xinkun Wang, Ching Man Wai, Jyothi Thimmapuram, Devender Arora, Tania Mesa, Jun Fan, Yuriy O. Alekseyev, Felice Cervone, Christopher Williams, Nickolas G. Gorham, Alexander Lemenze, Sara Goodwin, Jonathan Preall, Charles A. WhittakerList of authors in order
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https://doi.org/10.1101/2025.10.24.684427Publisher landing page
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YesWhether a free full text is available
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greenOpen access status per OpenAlex
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https://doi.org/10.1101/2025.10.24.684427Direct OA link when available
- Cited by
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0Total citation count in OpenAlex
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38Number of works referenced by this work
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