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. Sample of WT mouse dura mater (pooled from 6 mice)脑-免疫串扰（Brain-immune crosstalk）与神经炎症在生理与病理状态下均对大脑生理功能发挥关键调控作用。深入解析大脑免疫微环境，对于开发多种神经系统疾病的新型治疗手段至关重要。单细胞技术能够实现对免疫细胞异质性、动态变化与功能特征的无偏评估。本研究提供一套标准化实验方案，详细阐述大脑免疫区室的单细胞多组学分析所需的全部实验步骤。该方案包含小鼠大脑边界区域的显微解剖分步操作指南，以及针对各组织优化的细胞解离流程，可实现高细胞得率并最大限度减少解离过程诱导的基因表达变化。随后，本方案详述了基于液滴的单细胞RNA测序（scRNA-seq）、转录组与表位测序细胞索引技术（CITE-seq）以及高维流式细胞术的实验流程。上述各类单细胞检测技术各有优劣，但彼此间具有高度的互补性。尤为重要的是，本方案详细阐述了如何使用大规模抗体组合开展CITE-seq实验，从而实现无偏的蛋白质表达筛选与转录组分析的联合检测。最后，本方案详述了数据的分析与解读核心步骤。这套优化后的实验流程可在RNA与蛋白质层面，对全脑或特定边界区域的免疫细胞异质性与活化状态进行深入解析。湿实验（wetlab）流程可由经过专业培训、具备细胞与分子生物学基础操作能力的研究人员完成，总耗时6至11小时，具体时长取决于所选实验方案。后续的计算分析则需要研究者具备生物信息学与R语言编程基础。野生型（wild type, WT）小鼠硬脑膜样本（由6只小鼠样本混合制备）