title: NextSeq 500 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: EVs from rat AFSCs were isolated from cells that were treated with exosome-depleted FBS (ThermoFisher, Waltham, MA) for 18 hours by ultracentrifugation as previously described [Antounians et al, 2018] Treatment with EVs is with 10% (by volume) EVs were processed for total RNA isolation using the SeraMir Exosome RNA Purification Column kit (System Biosciences, Palo Alto, CA) according to the manufacturer's instructions. For each sample, 1μL of the final RNA eluate was used for measurement of small RNA concentration by Agilent Bioanalyzer Small RNA Assay using Bioanalyzer 2100 Expert instrument (Agilent Technologies, Santa Clara, CA). Small RNA libraries were constructed with the CleanTag Small RNA Library Preparation Kit (TriLink, San Diego, CA) according to the manufacturer's protocol. The final purified library was quantified with High Sensitivity DNA Reagents (Agilent Technologies, Santa Clara, CA) and High Sensitivity DNA Chips (Agilent Technologies, Santa Clara, CA). The libraries were pooled, and the 140bp to 300bp region was size selected on an 8% TBE gel (Invitrogen, Carlsbad, CA). The size selected library is quantified with High Sensitivity DNA 1000 Screen Tape (Agilent Technologies, Carlsbad, CA), High Sensitivity D1000 reagents (Agilent Technologies, Carlsbad, CA), and the TailorMix HT1 qPCR assay (SeqMatic, Fremont, CA).