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

Janilyn Arsenio

Methods in molecular biology (Clifton, N.J.) 2020

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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.

Citation

Janilyn Arsenio. Single-Cell Transcriptomics of Immune Cells: Cell Isolation and cDNA Library Generation for scRNA-Seq. Methods in molecular biology (Clifton, N.J.). 2020;2184:1-18