Rapid and robust isolation of microglia and vascular cells from brain subregions for integrative single-cell analyses

Cell isolation protocols from brain tissue constitute prolonged ex vivo processing durations, rendering them suboptimal for transcriptomic studies. specially, yielding sufficient quantities of microglia or vascular cells requires the use of large tissue volumes and the addition of an enrichment step. In this study, we developed a simple, rapid, and reproducible cell isolation method for generation of single-cell suspensions from micro-dissected brain regions, providing high numbers of microglia and vascular cells, without requiring an additional encrihment step. Cells isolated using this method are suitable for molecular profiling studies using 10 Genomics Chromium single-cell RNA sequencing with high reproducibility. Our method is valuable for longitudinal unbiased molecular profiling of microglia and vascular cells within different brain regions, spanning multiple time points across physiological development or disease progression. Female WT C57Bl/6J (Charles River, Sulzfeld, Germany, stock #000664) were deeply anesthetized with sodium pentobarbital (100 mg/kg, ABCUR AB #444362, Sweden) and transcardially perfused with 20 ml of ice-cold 1 phosphate buffered saline without Ca2+ and Mg2+ (PBS; pH 7.4; Gibco/Life Technologies #10010056). Brain cerebral cortices were isolated from three three-week-old and hippocampi were isolated from three five-week-old mice. Cells were isolated per each cortical or hippocampal preparation. Tissues from all 3 experiments were handled in the same way for the single-cell suspension. Isolated cells were processed for single-cell RNA sequencing at the eukaryotic single-cell genomics facility (ESCG) at Karolinska Institutet, Sweden using the 10x Genomics Chromium method (version 3.0.1) and sequenced using Illumina NovaSeq 600 with an S2 flow cell. The Cellranger 5.0.1 pipeline was used to align the raw sequencing reads to the mouse reference genome, Mus musculus version mm10, and generate the unique molecular identifier (UMI) count matrix. Each preparation included 3 technical replicates and each replicate was processed independently before integrating them together.脑组织细胞分离方案通常需要较长的离体处理时长，因此其并不适配转录组学研究的需求。具体而言，若要获取足量的小胶质细胞或血管细胞，需使用较大体积的脑组织样本，并额外增设富集步骤。 本研究开发了一种简便、快速且可重复的细胞分离方法，可从显微解剖的脑组织区域制备单细胞悬液，无需额外富集步骤即可获得大量小胶质细胞与血管细胞。采用本方法分离得到的细胞可适配10x Genomics Chromium单细胞RNA测序的分子谱分析研究，且重现性极佳。 本方法可用于不同脑区中小胶质细胞与血管细胞的纵向无偏分子谱分析，覆盖生理发育或疾病进程中的多个时间节点。 选取雌性野生型C57Bl/6J小鼠（Charles River，德国祖尔茨菲尔德，货号#000664），以戊巴比妥钠（100 mg/kg，ABCUR AB，瑞典，货号#444362）深度麻醉，随后经心脏灌注20 mL预冷的1×无钙镁磷酸盐缓冲液（PBS；pH 7.4；Gibco/Life Technologies，货号#10010056）。 从3只3周龄小鼠体内分离大脑皮层，从3只5周龄小鼠体内分离海马组织。每份皮层或海马样本均单独进行细胞分离。所有3组实验的组织在制备单细胞悬液时均采用相同处理流程。 分离得到的细胞于瑞典卡罗林斯卡学院真核单细胞基因组学平台（eukaryotic single-cell genomics facility, ESCG）进行处理，采用10x Genomics Chromium试剂盒（版本3.0.1）进行单细胞RNA测序，并使用Illumina NovaSeq 600测序仪搭配S2流动槽完成测序。 使用Cellranger 5.0.1分析流程将原始测序读数比对至小鼠参考基因组（Mus musculus，版本mm10），并生成唯一分子标识符（unique molecular identifier, UMI）计数矩阵。 每份样本设置3个技术重复，所有重复均单独处理后再进行整合分析。