Preparation of single cells from tumors for single-cell RNA sequencing

Methods Enzymol. 2020;632:295-308. doi: 10.1016/bs.mie.2019.05.057. Epub 2019 Jun 18.


Intratumoral heterogeneity of cancer cells and tumor-infiltrating immune cells is increasingly being viewed as a key factor driving tumor progression and response to therapy. Over the past several years, technological advances have created powerful tools to analyze the tumor microenvironment on a single-cell level, including mass cytometry and single-cell RNA sequencing, which is particularly pertinent to tumor immunology and cancer immunotherapy. The integrity and reliability of the data generated from these single-cell technologies, however, are highly influenced by the process and quality of sample preparation, which, if carried out inappropriately, has a potential to produce misleading results. In this chapter, we describe a protocol for the generation of single cell suspensions from human tumor samples that has been optimized for single-cell RNA sequencing. This protocol can be easily adapted for other single cell applications such as mass and flow cytometry. Throughout the entire workflow, we aim to maximize viability and minimize factors contributing to cellular stress that could affect downstream analyses.

Keywords: Dissociation; Extracellular matrix; Flow cytometry; Immunology; Single cell; Single-cell RNAseq; Suspension.

MeSH terms

  • Cell Separation / methods
  • Humans
  • Neoplasms / genetics*
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*
  • Specimen Handling / methods
  • Workflow