Marsh, Samuel E;
Walker, Alec J;
Kamath, Tushar;
Dissing-Olesen, Lasse;
Hammond, Timothy R;
de Soysa, T Yvanka;
Young, Adam MH;
... Stevens, Beth; + view all
(2022)
Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.
Nature Neuroscience
, 25
(3)
pp. 306-316.
10.1038/s41593-022-01022-8.
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2020.12.03.408542v1.full.pdf - Accepted Version Download (7MB) | Preview |
Abstract
A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.
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