单细胞转录组学分析免疫细胞：单细胞 RNA 测序的免疫细胞分离和 cDNA 文库构建。

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

机构信息

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

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

出版信息

Methods Mol Biol. 2020;2184:1-18. doi: 10.1007/978-1-0716-0802-9_1.

DOI:10.1007/978-1-0716-0802-9_1

PMID:32808214

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.

摘要

单细胞 RNA 测序 (scRNA-seq) 通过下一代测序实现了对单个细胞转录组的全面分析。scRNA-seq 为研究不同生物系统（包括免疫系统）的细胞异质性和动态性提供了一种无偏倚的方法。在进行 RNA-seq 分析之前，对技术程序进行优化对于 scRNA-seq 实验的成功至关重要。在这里，描述了三个主要的实验程序：（1）从原代鼠组织中分离免疫 CD8a T 细胞，（2）使用 10× Genomics Chromium Controller 和 Chromium Single Cell 3' Solution 生成单细胞 cDNA 文库，以及（3）cDNA 文库质量控制。在本方案中，CD8a T 细胞从鼠脾组织中分离，但任何感兴趣的细胞类型都可以富集并用于单细胞 cDNA 文库的生成和随后的 RNA-seq 实验。