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Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows
BACKGROUND: Single-cell RNA sequencing has been widely adopted to estimate the cellular composition of heterogeneous tissues and obtain transcriptional profiles of individual cells. Multiple approaches for optimal sample dissociation and storage of single cells have been proposed as have single-nucl...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265231/ https://www.ncbi.nlm.nih.gov/pubmed/32487174 http://dx.doi.org/10.1186/s13059-020-02048-6 |
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author | Denisenko, Elena Guo, Belinda B. Jones, Matthew Hou, Rui de Kock, Leanne Lassmann, Timo Poppe, Daniel Clément, Olivier Simmons, Rebecca K. Lister, Ryan Forrest, Alistair R. R. |
author_facet | Denisenko, Elena Guo, Belinda B. Jones, Matthew Hou, Rui de Kock, Leanne Lassmann, Timo Poppe, Daniel Clément, Olivier Simmons, Rebecca K. Lister, Ryan Forrest, Alistair R. R. |
author_sort | Denisenko, Elena |
collection | PubMed |
description | BACKGROUND: Single-cell RNA sequencing has been widely adopted to estimate the cellular composition of heterogeneous tissues and obtain transcriptional profiles of individual cells. Multiple approaches for optimal sample dissociation and storage of single cells have been proposed as have single-nuclei profiling methods. What has been lacking is a systematic comparison of their relative biases and benefits. RESULTS: Here, we compare gene expression and cellular composition of single-cell suspensions prepared from adult mouse kidney using two tissue dissociation protocols. For each sample, we also compare fresh cells to cryopreserved and methanol-fixed cells. Lastly, we compare this single-cell data to that generated using three single-nucleus RNA sequencing workflows. Our data confirms prior reports that digestion on ice avoids the stress response observed with 37 °C dissociation. It also reveals cell types more abundant either in the cold or warm dissociations that may represent populations that require gentler or harsher conditions to be released intact. For cell storage, cryopreservation of dissociated cells results in a major loss of epithelial cell types; in contrast, methanol fixation maintains the cellular composition but suffers from ambient RNA leakage. Finally, cell type composition differences are observed between single-cell and single-nucleus RNA sequencing libraries. In particular, we note an underrepresentation of T, B, and NK lymphocytes in the single-nucleus libraries. CONCLUSIONS: Systematic comparison of recovered cell types and their transcriptional profiles across the workflows has highlighted protocol-specific biases and thus enables researchers starting single-cell experiments to make an informed choice. |
format | Online Article Text |
id | pubmed-7265231 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-72652312020-06-07 Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows Denisenko, Elena Guo, Belinda B. Jones, Matthew Hou, Rui de Kock, Leanne Lassmann, Timo Poppe, Daniel Clément, Olivier Simmons, Rebecca K. Lister, Ryan Forrest, Alistair R. R. Genome Biol Research BACKGROUND: Single-cell RNA sequencing has been widely adopted to estimate the cellular composition of heterogeneous tissues and obtain transcriptional profiles of individual cells. Multiple approaches for optimal sample dissociation and storage of single cells have been proposed as have single-nuclei profiling methods. What has been lacking is a systematic comparison of their relative biases and benefits. RESULTS: Here, we compare gene expression and cellular composition of single-cell suspensions prepared from adult mouse kidney using two tissue dissociation protocols. For each sample, we also compare fresh cells to cryopreserved and methanol-fixed cells. Lastly, we compare this single-cell data to that generated using three single-nucleus RNA sequencing workflows. Our data confirms prior reports that digestion on ice avoids the stress response observed with 37 °C dissociation. It also reveals cell types more abundant either in the cold or warm dissociations that may represent populations that require gentler or harsher conditions to be released intact. For cell storage, cryopreservation of dissociated cells results in a major loss of epithelial cell types; in contrast, methanol fixation maintains the cellular composition but suffers from ambient RNA leakage. Finally, cell type composition differences are observed between single-cell and single-nucleus RNA sequencing libraries. In particular, we note an underrepresentation of T, B, and NK lymphocytes in the single-nucleus libraries. CONCLUSIONS: Systematic comparison of recovered cell types and their transcriptional profiles across the workflows has highlighted protocol-specific biases and thus enables researchers starting single-cell experiments to make an informed choice. BioMed Central 2020-06-02 /pmc/articles/PMC7265231/ /pubmed/32487174 http://dx.doi.org/10.1186/s13059-020-02048-6 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Denisenko, Elena Guo, Belinda B. Jones, Matthew Hou, Rui de Kock, Leanne Lassmann, Timo Poppe, Daniel Clément, Olivier Simmons, Rebecca K. Lister, Ryan Forrest, Alistair R. R. Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows |
title | Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows |
title_full | Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows |
title_fullStr | Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows |
title_full_unstemmed | Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows |
title_short | Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows |
title_sort | systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus rna-seq workflows |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265231/ https://www.ncbi.nlm.nih.gov/pubmed/32487174 http://dx.doi.org/10.1186/s13059-020-02048-6 |
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