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. 2021 Jun 10;2(2):100590.
doi: 10.1016/j.xpro.2021.100590. eCollection 2021 Jun 18.

Dissociation of microdissected mouse brain tissue for artifact free single-cell RNA sequencing

Lu Liu  1 , Simon Besson-Girard  1 , Hao Ji  1 , Katrin Gehring  1 , Buket Bulut  1 , Tuğberk Kaya  1   2   3 , Fumere Usifo  1 , Mikael Simons  4   2   3 , Ozgun Gokce  1   4
Affiliations

Dissociation of microdissected mouse brain tissue for artifact free single-cell RNA sequencing

Lu Liu et al. STAR Protoc. .

Abstract

Single-cell RNA sequencing (scRNA-seq) provides the transcriptome of individual cells and addresses previously intractable problems including the central nervous system's transcriptional responses during health and disease. However, dissociating brain cells is challenging and induces artificial transcriptional responses. Here, we describe an enzymatic dissociation method for mouse brain that prevents dissociation artifacts and lowers technical variations with standardized steps. We tested this protocol on microdissected brain tissue of 3-week- to 24-month-old mice and obtained high-quality scRNA-seq results. For complete details on the use and execution of this protocol, please refer to Safaiyan et al. (2021).

Keywords: Cell Biology; Cell isolation; Flow Cytometry/Mass Cytometry; Neuroscience; RNA-seq; Sequencing; Single Cell.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Workflow Graphic summary of the main steps leading to different single cell library preparation methods
Figure 2
Figure 2
Images of preparation (A) Images of the set up before the experiment: the tubes harboring the prepared enzyme mixture. (B) Images of the set up for dissecting brain tissue. (C) Close up of the set up for dissecting mouse brain tissue.
Figure 3
Figure 3
Images of critical steps (A) Dissociation: Images of the C tube attached to the gentleMACS™ Octo Dissociator with Heaters. (B) Debris removal: Images of the two layers (layer1+layer2) to be removed after first centrifugation in the debris removal process. (C) Images of the cell suspension before and after myelin removal process. (D) Myelin removal: Images of the set up for the myelin removal process.
Figure 4
Figure 4
Images on using the flow cytometry cell strainer to filter low volume cell suspension
Figure 5
Figure 5
Full gating strategy for the selection of single live (7AAD-) microglia (CD45+CD11b+) of white matter from a 24-month-old mouse brain
See this image and copyright information in PMC

References

    1. Bordt E.A., Block C.L., Petrozziello T., Sadri-Vakili G., Smith C.J., Edlow A.G., Bilbo S.D. Isolation of microglia from mouse or human tissue. STAR Protoc. 2020;1:100035. - PMC - PubMed
    1. Gehring J., Park J.H., Chen S., Thomson M., Pachter L. Highly multiplexed single-cell RNA-seq by DNA oligonucleotide tagging of cellular proteins. Nat. Biotechnol. 2020;38:35–38. - PubMed
    1. Nicolás-Ávila J.Á., Sanchez-Diaz M., Hidalgo A. Isolation of exophers from cardiomyocyte-reporter mouse strains by fluorescence-activated cell sorting. STAR Protoc. 2021;2:100286. - PMC - PubMed
    1. Safaiyan S., Besson-Girard S., Kaya T., Cantuti-Castelvetri L., Liu L., Ji H., Schifferer M., Gouna G., Usifo F., Kannaiyan N. White matter aging drives microglial diversity. Neuron. 2021;109:1100–1117. - PubMed
    1. Stoeckius M., Zheng S., Houck-Loomis B., Hao S., Yeung B.Z., Mauck W.M., Smibert P., Satija R. Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 2018;19:1–12. - PMC - PubMed

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