Illumina HiSeq 1500 paired end sequencing; Study of human macrophage reprogramming toward a pro-resolving profile by efferocytosis
Leukocyte-platelet concentrates and cytapheresis kits were obtained from healthy volunteers after informed consent signature. Samples were collected from the French Blood Transfusion Center EFS Bourgogne Franche-Comté, Besançon, France). Human primary neutrophils were isolated at room temperature from fresh leukocyte-platelet concentrates using a Ficoll-density gradient (Eurobio) followed by sedimentation in a 3% dextran solution (Sigma). Red blood cells were lysed by 5 min incubation in hypertonic solution (NH4Cl (0.077 M), EDTA (0.633 mM), KHCO3 (5 mM)). Neutrophils were resuspended in RPMI 1640-GlutaMAX-HEPES (Gibco) and incubated at 37°C, 5% CO2. Neutrophils were further handled carefully to avoid any mechanical stress. Human monocytes were isolated from fresh cytapheresis kit using a Ficoll-density gradient followed by positive CD14+ selection (human CD14+ MicroBeads, Miltenyi). Purity was assessed by CD14 labelling by flow cytometry. Monocytes were resuspended in RPMI 1640-GlutaMAX(TM)-HEPES (Gibco) with M-CSF, or M-CSF + IL4 , or GM-CSF + IFN (Miltenyi). Every cytokines were used at 50 ng/mL. Cells were kept at 37°C, 5% CO2 for 7 days to allow differentiation into macrophages and one volume of medium was added at day 3. For mixing experiments, neutrophils were harvested, resuspended in X-Vivo15 (Gibco) and mixed with macrophages at a 5:1 ratio for the indicated period. Remaining neutrophils were then removed by extensive PBS washes, and macrophages were harvested by scrapping in a PBS-10mM EDTA solution and further processed. RNA was extracted with NucleoSpin® RNA Plus kit (Macherey-Nagel) according to manufacturer's instructions. RNA quantity was measured using a Nanodrop spectrophotometer (Thermo-Fisher). RNA quality was assessed using an Experion™ Automated Electrophoresis System according to manufacturer's instructions. Libraries were prepared from 200 ng of RNAs with the Poly(A) mRNA Magnetic Isolation Module (Biolabs) and NEBNext Ultra II Directional RNA Library Preparation Kit (Biolabs)