Single-Cell Transcriptomics of Immune Cells: Cell Isolation and cDNA Library Generation for scRNA-Seq.
Methods Mol Biol
; 2184: 1-18, 2020.
Article
in En
| MEDLINE
| ID: mdl-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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA
/
Sequence Analysis, RNA
/
CD8-Positive T-Lymphocytes
/
RNA, Small Cytoplasmic
/
Gene Expression Profiling
/
Single-Cell Analysis
/
Transcriptome
Limits:
Animals
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2020
Document type:
Article
Affiliation country:
Canadá