Illumina HiSeq 4000 sequencing; Quiescent endothelial cells upregulate fatty acid β-oxidation for vasculoprotection via redox homeostasis
Quiescent endothelial cells upregulate fatty acid β-oxidation for vasculoprotection via redox homeostasis
Mouse endothelial cells were isolated from perfused healthy lungs of CPT1A∆EC mice and their wild-type littermates. Briefly, before starting the perfusion, the mice were anesthetized with Ketamin/Xylasine (500μL/mouse of Ketamine 0.65% / Xylasine 0.05% diluted in saline solution)   and once the withdrawal reflex was absent in pelvic limbs, the perfusion procedure was started. Mice were perfused with 5 ml of a water based perfusion buffer containing 1.7 M NaCl, 84 mM KCl, 120 mM Hepes and 1 mM NaOH followed by perfusion with 5 ml of a DMEM based digestion buffer containing 0.1% collagenase II, collagenase I, 2 mM CaCl2, 1% antibiotic/antimycotic, at a perfusion rate of 2 ml/min. Lungs were dissected and further dissociated using the gentleMACS dissociator system. Next, samples were centrifuged, resuspended in 5ml of PBS/0.5% BSA and applied to a pre-separation 70 μm filter. The filtered cells were washed twice with PBS/BSA buffer and endothelial cells were selected with CD31 MicroBeads (Milenyi), further stained with viability dye, CD102-APC and CD45-PeCy7 and the fraction of viable, CD102+ and CD45- cells obtained by FACS sorting was directly collected into Trizol reagent. RNA was extracted using TRIzol (Life Technologies). Starting from 1 µg total RNA, poly-adenylated fragments were isolated, reverse transcribed and converted into indexed sequencing libraries using the KAPA stranded mRNA-seq kit (Sopachem, Eke, Belgium) RNA libraries were created using the KAPA stranded mRNA-seq kit (Sopachem, Eke, Belgium) according to the manufacturers instructions.
Experimental Factor: phenotype
wild type