RNA-seq of human type 2 innate lymphoid cells (ILC2) stimulated with PGD2 or PGD2 metabolites (Δ12PGJ2, Δ12PGD2, 15-deoxyΔ12,14PGD2, 9α,11β-PGF2) with or without the selective DP2 antagonist fevipiprant
RNA-seq of human type 2 innate lymphoid cells (ILC2) stimulated with PGD2 or PGD2 metabolites (Δ12PGJ2, Δ12PGD2, 15-deoxyΔ12,14PGD2, 9α,11β-PGF2) with or without the selective DP2 antagonist fevipiprant
Peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL peripheral blood of patients with atopic asthma (n = 4) using Ficoll density gradient centrifugation. ILC2 were enriched by depletion of T cells, B cells and monocytes using CD3-, CD14- and CD19-MACS separation beads (Miltenyi Biotech, Bergisch Gladbach, Germany), respectively. ILC2 cells (CD45+, CD4-, CD8-, CD14-, CD16-, CD19-, CD34-, CD123-, FcεRI-, CD11b-, CD56-, CD3-, CD127+, CD294+) were sorted with an FACS ARIA Fusion (BD Bioscience) into 96 U bottom well plates (100 cells/ well). Sorted cells were expanded with human feeder PBMCs (100,000/ well; 37°C, 5% CO2) in culture medium (RPMI 1640 Glutamax medium, 1% Pen/Strep, 10% h.i. human AB serum, 25 mM HEPES, 100 U/ ml rh-IL-2, 25 ng/ ml rh-IL-4, and 5 µg/ ml phytohemagglutinin-M (PHA M)) for three to five weeks. Cells (≤ 31,000-150,000/ well  ) were incubated with 1 µM fevipiprant for 1 h and afterwards with the EC70 concentration of PGD2 or selected PGD2 metabolites (Δ12-PGD2, Δ12-PGJ2, 15-deoxy-Δ12,14-PGD2, 9α,11β-PGF2) metabolites for 24 h (U-bottom 96-well plates, 37°C, 5% CO2). After centrifugation (5 min, 300 x g, RT), cell samples were stabilized in RNAprotect Cell Reagent (Qiagen, Venlo, The Netherlands) and stored at -80°C until further handling.  Total RNA was isolated from ILC2 using the RNeasy® Plus Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. RNA was eluted with RNase-free water and the concentration as well as the purity were determined via absorbance measurement using a NanoDrop device. Eluted RNA was stored at -80°C.  Library preparation was performed using the NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Frankfurt am Main, Germany) following the manufacturer's instructions.