Single-Cell Gene Expression, Clonality, and Feature Barcoding of Melanoma Tumor-Infiltrating Lymphocytes.
Angela Pizzolla, Simon P Keam, Criselle D'Souza, Timothy Semple, Paul J Neeson
Methods Mol Biol | Published : 2021
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..View full abstract