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Immunometabolism pp 1–18Cite as

Single-Cell Transcriptomics of Immune Cells: Cell Isolation and cDNA Library Generation for scRNA-Seq

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2184))

Abstract

Single-cell RNA-sequencing (scRNA-seq) enables a comprehensive analysis of the transcriptome of individual cells by next-generation sequencing. ScRNA-seq offers an unbiased approach to investigate the cellular heterogeneity and dynamics of diverse biological systems, including the immune system. Optimization of the technical procedures performed prior to RNA-seq analysis is imperative to the success of a scRNA-seq experiment. Here, three major experimental procedures are described: (1) the isolation of immune CD8a+ T cells from primary murine tissue, (2) the generation of single-cell cDNA libraries using the 10× Genomics Chromium Controller and the Chromium Single Cell 3′ Solution, and (3) cDNA library quality control. In this protocol, CD8a+ T cells are isolated from murine spleen tissue, but any cell type of interest can be enriched and used for single-cell cDNA library generation and subsequent RNA-seq experiments.

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Acknowledgments

This work was supported by the Canada Research Chairs Program, Canadian Foundation for Innovation John R. Evans Leaders Fund, Research Manitoba, Health Sciences Centre Foundation, Natural Sciences and Engineering Council of Canada Discovery Grant, and the Department of Internal Medicine, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba.

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Authors and Affiliations

  1. Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

    Janilyn Arsenio

  2. Manitoba Centre for Proteomics and Systems Biology, Winnipeg, Manitoba, Canada

    Janilyn Arsenio

  3. Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada

    Janilyn Arsenio

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  1. Janilyn Arsenio
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Correspondence to Janilyn Arsenio .

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Editors and Affiliations

  1. Faculty of Health Sciences, Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada

    Suresh Mishra

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Arsenio, J. (2020). Single-Cell Transcriptomics of Immune Cells: Cell Isolation and cDNA Library Generation for scRNA-Seq. In: Mishra, S. (eds) Immunometabolism. Methods in Molecular Biology, vol 2184. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0802-9_1

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  • DOI: https://doi.org/10.1007/978-1-0716-0802-9_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0801-2

  • Online ISBN: 978-1-0716-0802-9

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