title: GSM9294013: Cortex Ctrl Stroke; Mus musculus; RNA-Seq
library_construction_protocol: Brain tissue was dissociated with the Miltenyi neural dissociation kit following the manufacturer's instructions, with minor adjustments to optimize the isolation of endothelial and immune cells. In brief, 4–5 mice per treatment group were anesthetized with ketamine/xylazine solution and perfused with 15mL ice cold phosphate buffered saline (PBS) following cardiac blood collection. The stroke core and peri-infarct cortex, defined as ~2mm surrounding the visible lesion, were dissected and pooled by condition, minced, and digested using the neural dissociation kit (Miltenyi, 130–092-628). Brain homogenates were further diluted in Dulbecco's PBS (dPBS) and filtered through a 100 µm strainer. The filtered homogenate was then centrifuged (300 g for 10 minutes) and the pellets were resuspended in 0.9 M sucrose in dPBS followed by centrifugation for 20 minutes at 850 g at 4 °C to separate the myelin. Cell pellets were resuspended in FACS buffer (1% BSA in PBS with 2 mM EDTA) and blocked for 5 min on ice with Fc block (CD16/CD32, BD 553141), followed by staining for 10 minutes with anti-CD31-PE/TR594 (1:200, BD 563616) and anti-CD45-PE/Cy7 (1:200, BD 103114) antibodies. Dead cells were excluded by staining with SYTOX Blue Dead Cell Stain (1:5,000, Thermo Fisher Scientific S34857). Reagents of the Chromium Single Cell 3' GEM & Gel Bead Kit v3.1 (10X Genomics, 1000121) were thawed and prepared according to the manufacturer's protocol. Cells and master mix solution were adjusted to target 10,000 cells per sample and loaded on a standard Chromium Controller (10X Genomics, 1000204) according to manufacturer protocols. We applied 11 PCR cycles to generate cDNA. Library construction was conducted using the Chromium Single Cell 3' Library Construction Kit v3 (10X Genomics, 1000121). All reaction and quality control steps were carried out according to the manufacturer's protocol and with recommended reagents, consumables, and instruments. Quality control of cDNA and libraries was conducted using a Bioanalyzer (Agilent) at the Stanford Protein and Nucleic Acid Facility. Illumina sequencing of the resulting libraries was performed by Novogene (https://en.novogene.com/) on an Illumina NovaSeq S4 (Illumina). Base calling, demultiplexing, and generation of FastQ files were conducted by Novogene.