Single-cell RNA and protein profiling of immune cells from the mouse brain and its border tissues

Nat Protoc. 2022 Oct;17(10):2354-2388. doi: 10.1038/s41596-022-00716-4. Epub 2022 Aug 5.

Abstract

Brain-immune cross-talk and neuroinflammation critically shape brain physiology in health and disease. A detailed understanding of the brain immune landscape is essential for developing new treatments for neurological disorders. Single-cell technologies offer an unbiased assessment of the heterogeneity, dynamics and functions of immune cells. Here we provide a protocol that outlines all the steps involved in performing single-cell multi-omic analysis of the brain immune compartment. This includes a step-by-step description on how to microdissect the border regions of the mouse brain, together with dissociation protocols tailored to each of these tissues. These combine a high yield with minimal dissociation-induced gene expression changes. Next, we outline the steps involved for high-dimensional flow cytometry and droplet-based single-cell RNA sequencing via the 10x Genomics platform, which can be combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and offers a higher throughput than plate-based methods. Importantly, we detail how to implement CITE-seq with large antibody panels to obtain unbiased protein-expression screening coupled to transcriptome analysis. Finally, we describe the main steps involved in the analysis and interpretation of the data. This optimized workflow allows for a detailed assessment of immune cell heterogeneity and activation in the whole brain or specific border regions, at RNA and protein level. The wet lab workflow can be completed by properly trained researchers (with basic proficiency in cell and molecular biology) and takes between 6 and 11 h, depending on the chosen procedures. The computational analysis requires a background in bioinformatics and programming in R.

Publication types

  • Review

MeSH terms

  • Animals
  • Brain
  • Epitopes
  • Gene Expression Profiling / methods
  • High-Throughput Nucleotide Sequencing* / methods
  • Mice
  • RNA* / genetics
  • Sequence Analysis, RNA / methods
  • Single-Cell Analysis / methods
  • Transcriptome

Substances

  • Epitopes
  • RNA