RNA-Seq of mouse pancreatic islets treated with cytokines and NAMPT inhibitor (FK866)
RNA-Seq of mouse pancreatic islets treated with cytokines and NAMPT inhibitor (FK866)
Mouse islets were isolated between 10:00 and 11:00 hours. Pancreata were inflated with 1mg/mL collagenase solution (Sigma-Aldrich, Poole, U.K.) followed by density gradient separation (Histopaque-1077; Sigma-Aldrich). Isolated islets were maintained in supplemented RPMI at 37 °C and 5% CO2 in a humidified atmosphere for up to 24 hours prior to experimental treatment. Isolated mouse islets were subjected for 24 h to (A) no treatment, (B) a cytokine cocktail (IL-1β, TNFα and IFNγ; 1 ng/ml) or (C) FK866 (10 nM) and cytokine cocktail. Total RNA was extracted from ∼150 islets per mouse, according to the manufacturer's instructions (RNeasy Mini Kit, Qiagen, UK) 100ng total RNA from each sample was used as input to generate library for RNAseq. RNAseq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep kit (New England Biolabs, Hertfordshire, UK. Cat.no: E7760) following manufacturer's recommendations.