title: Illumina NovaSeq 6000 paired end sequencing; Impaired Fetal Lung Development can be Rescued by Administration of Extracellular Vesicles Derived from Amniotic Fluid Stem Cells
design: Impaired Fetal Lung Development can be Rescued by Administration of Extracellular Vesicles Derived from Amniotic Fluid Stem Cells
library_construction_protocol: 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. We used an automated NEBNext Ultra II Directional with polyA isolation (New England BioLabs) using the Agilent NGS Workstation (Agilent Technologies) as per manufacturer's protocol. Briefly, 250 ng of total RNA spiked-in with ERCCs (External RNA Controls Consortium, Ambion) as per manufacturer's protocol was used to generate cDNA.