Isolation of myeloid cells from mouse brain tumors for single-cell RNA-seq analysis

Mai T Dang; Fernanda Mafra; Malay Haldar

doi:10.1016/j.xpro.2021.100957

Isolation of myeloid cells from mouse brain tumors for single-cell RNA-seq analysis

STAR Protoc. 2021 Nov 17;2(4):100957. doi: 10.1016/j.xpro.2021.100957. eCollection 2021 Dec 17.

Authors

Mai T Dang^{1

2}, Fernanda Mafra³, Malay Haldar^{4

5

6}

Affiliations

¹ Division of Neurology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
² Division of Pediatric and Developmental Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.
³ Center for Applied Genomics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
⁴ Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁵ Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁶ Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Abstract

Current single-cell RNA sequencing (scRNA-seq) protocols are limited by the number of cells that can be simultaneously sequenced, restricting the ability to resolve heterogeneity of rare cell types. We describe here a protocol for rapid isolation of myeloid cells from tumor-harboring mouse cerebellum without cell sorting to minimize cell damage for scRNA-seq. This protocol includes the procedures for further enrichment of myeloid cells using CD11b+ magnetic beads, followed by the generation of scRNA library and sequencing analysis. For complete details on the use and execution of this protocol, please refer to Dang et al. (2021).

Keywords: Cancer; Cell Biology; Cell isolation; Immunology; RNAseq; Sequencing; Single Cell.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Brain / cytology
Brain Neoplasms / pathology*
Cell Separation / methods*
Male
Mice
Mice, Transgenic
Myeloid Cells / cytology*
Sequence Analysis, RNA / methods*
Single-Cell Analysis / methods*

Abstract

Publication types

MeSH terms

Grants and funding