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. Overall design: 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.