Illumina HiSeq 2500 sequencing; Impaired Fetal Lung Development can be Rescued by Administration of Extracellular Vesicles Derived from Amniotic Fluid Stem Cells
Impaired Fetal Lung Development can be Rescued by Administration of Extracellular Vesicles Derived from Amniotic Fluid Stem Cells
At E14.5, a single cell suspension was obtained from pooled lungs from either control or nitrofen exposed rat fetuses by trypsinization (0.25% Trypsin-EDTA, ThermoFisher, Waltham, MA) for 20 minutes at 37°C humidified chamber. Cells were spun down by centrifugation for 5 minutes at 800g and the pellet was resuspended in DMEM supplemented with 10% FBS and subjected to three serial depletion of fibroblasts by incubation for 1 hour each following an established protocol [Caniggia 1991]. Groups were assigned randomly. Treatment with EVs is with 10% (by volume) Total RNA was extracted using Nucleospin miRNA kit (Macherey-Nagel, Düren, Germany), following supplier recommended protocols. NEBNext small RNA kit was used to construct libraries.