GSE191075
Single-cell RNA and protein profiling of immune cells from the mouse brain and its border tissues
Brain-immune crosstalk 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 many 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 for performing single-cell multi-omic analysis of the brain immune compartment. This includes a step-by-step description on how to micro-dissect 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 droplet-based single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), or high-dimensional flow cytometry. Each of these single-cell modalities has specific strengths and limitations, but they are highly complementary. 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 wetlab workflow can be completed by properly trained researchers (with basic proficiency in cell and molecular biology) and takes between 6 and 11 hours, depending on the chosen procedures. The computational analysis requires a background in bioinformatics and programming in R. Overall design: Sample of WT mouse dura mater (pooled from 6 mice)