RNA profiling of lung endothelial cells of mice under normal and high fat diet (HFD).

In order to analyze the genes and molecular pathways modulated by diet in the lungs of mice under DIO model, we performed RNA-seq on CD45-, CD31+ lung endothelial cells from mice under HFD, and compared their expression profiles with those under control diet. Lungs from mice under ND and HFD diet (12 weeks) were excised, disaggregated and digested in collagenase for 1 h at 37°C. The cell suspensions were seeded onto plates coated with fibronectin, collagen I and gelatin. After attachment, cells were negatively selected with anti-CD16/CD32 mAb coupled to magnetic beads, and then positively selected with anti-ICAM-2 coupled to magnetic beads. Cells were seeded onto plates coated with collagen I before RNA extraction. RNA was extracted with trizol and subsequent Qiagen columns. RNA sequencing was performed afterwards.