title: Alternative splicing and gene expression patterns of fetal cord-blood derived Endothelial Colony Forming Cells (ECFCs) in normal and preeclamptic pregnancies
library_construction_protocol: We centrifuged umbilical cord blood and separated plasma for storage at -80°C. After density gradient centrifugation, the cord blood mononuclear cell fraction was plated at 5 x 107 cells/well on collagen coated 6-well plates ((BioCoat; Corning, USA) and incubated at 37 °C and 5 % CO2. Cells were grown with endothelial basal medium (EBM-2, Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Harvard Bioscience, Harvard Bioscience, Holliston, USA), 1% penicillin/streptomycin (P/S, Bio&Sell, Nürnberg, Germany) and recommended supplements. After 10 to 21 days, ECFC colonies appeared as adherent single layers of cobblestone-shaped cells. ECFC phenotype was confirmed by flow cytometry assessing CD31 (130-117-390; BD Biosciences San Jose, USA), CD45 (555483; BD Biosciences), CD133 (130-090-826; Miltenyi Biotec, Bergisch Gladbach, Germany) and the corresponding isotype controls. Total RNA was purified from ECFC lysates using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). rRNA was depleted and PolyA enriched without globin depletion. A strand-specific library was utilized with the protocol NEB Ultra Directional RNA.