GSM8002103: M9, IRI; Mus musculus; RNA-Seq
For intravascular leukocyte staining, a PE-labeled rat anti-mouse CD45.2 antibody (2µg) was injected intravenously via the vena cava of anesthezised mice. Subsequently, puncture site was covered with surgical hemostat to control venous hemorrhage. After 5 minutes of antibody incubation blood was collected by cardiac puncture and organs were harvested without perfusion. Heparinzed blood was incubated with Fc Block (CD16/32) for 10 min on ice followed by antibody staining for 30 min. Erythrocytes were lysed for 10 min at RT using 1x RBC Lysis/Fixation solution (Biolegend). Samples were washed with PBS at 400 xg for 5 min and cell pellets were resuspended in PBS with 1% FCS. Organs were minced into small pieces and kidney, liver and lung were digested in 1 mL HBSS (Ca/Mg) including DNAse I (10mg/mL) and Collagenase I (100 U/µL) at 37°C and 350 rpm for 30 min (kidney) or 45 min (liver and lung). Addition of 2ml PBS to all organs was followed by filtering suspensions trough a 70 µm cell strainer. Samples were then centrifuged at 400 xg for 5 min and cell pellets were resuspended in PBS with 1% FCS. Cells were incubated with antibody mixtures (1:100) for 30 min on ice. Red blood cell lysis was performed for spleen according to blood while other organs were fixed with 4% PFA for 10 min. Cells were washed with 2 ml PBS (400 xg, 5 min) and resuspended in PBS with 1% FCS. Approximately 10,000-25,000 vital cells of each sample were processed for scRNA-seq using using BD Rhapsody Targeted mRNA & AbSeq Amplification Kit (BD Biosciences, Cat. No. 633774) according to the manufacturer's instructions. Quality, purity, average size and concentration of copy DNAs and libraries were determined by measurements on a Qubit FLuorometer (Thermo Fisher Scientific) and a Tapestation 2200 (Agilent Technologies). Indexed libraries were sequenced using a NovaSeq 6000 sequencing platform with PE150. flow cell and reagents (Illumina).