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Melanoma pp 529–541Cite as

Single-Cell Gene Expression, Clonality, and Feature Barcoding of Melanoma Tumor-Infiltrating Lymphocytes

Part of the Methods in Molecular Biology book series (MIMB,volume 2265)

Abstract

We describe here a protocol to measure gene expression, T cell receptor (TCR) sequence, and protein expression by single T cells extracted from melanoma, using 10× Chromium technology. This method includes freezing and thawing of the melanoma infiltrating lymphocytes, staining of cells with fluorescent and barcode-conjugated antibodies, sorting of T cells, and loading the cells on the 10× Chromium Controller. After sequencing, analysis includes quality control, genetic demultiplexing to resolve genetically different samples, and T cell clonality and clustering analysis. Single cell RNA sequencing paints the complete portrait of individual T cells, including their clonality and phenotype, and it reconstructs a complete picture of the T cell infiltrate in a tumor that is represented as cell clustering similar to a pointillism painting.

Key words

  • RNA
  • TCR
  • Sequencing
  • T cells
  • Antibodies
  • Clonality

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  • DOI: 10.1007/978-1-0716-1205-7_37
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References

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Author information

Authors and Affiliations

  1. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

    Angela Pizzolla, Simon P. Keam, Criselle D’Souza & Paul J. Neeson

  2. Molecular Genomics Core, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

    Timothy Semple

  3. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia

    Paul J. Neeson

Authors
  1. Angela Pizzolla
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  2. Simon P. Keam
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  3. Criselle D’Souza
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  4. Timothy Semple
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  5. Paul J. Neeson
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Corresponding author

Correspondence to Paul J. Neeson .

Editor information

Editors and Affiliations

  1. Department of Biology, Hampden-Sydney College, Hampden-Sydney, VA, USA

    Ph.D. Kristian M. Hargadon

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Pizzolla, A., Keam, S.P., D’Souza, C., Semple, T., Neeson, P.J. (2021). Single-Cell Gene Expression, Clonality, and Feature Barcoding of Melanoma Tumor-Infiltrating Lymphocytes. In: Hargadon, K.M. (eds) Melanoma. Methods in Molecular Biology, vol 2265. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1205-7_37

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  • DOI: https://doi.org/10.1007/978-1-0716-1205-7_37

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1204-0

  • Online ISBN: 978-1-0716-1205-7

  • eBook Packages: Springer Protocols