NextSeq 500 paired end sequencing; Extracellular Vesicles Derived from Amniotic Fluid Stem Cells Rescue Impaired Fetal Lung Development via the Release of Small RNAs
Extracellular Vesicles Derived from Amniotic Fluid Stem Cells Rescue Impaired Fetal Lung Development via the Release of Small RNAs
Rat amniotic fluid stem cells (AFSCs) were isolated from amniotic fluid of E12 rat fetuses as previously described [De Coppi 2007], and grown in alpha-minimal Essential Media (αMEM, Gibco, ThermoFisher, Waltham, MA) supplemented with 20% Chang supplements (Irvine Scientific, Santa Ana, CA), 15% fetal bovine serum (FBS, ThermoFisher Scientific, Waltham, MA), and 0.5% Penicillin/Streptomycin (ThermoFisher Scientific, Waltham, MA). Rat bone-marrow derived mesenchymal stem cells (MSCs) were purchased (CellBiologics, Chicago, IL) and grown in supplier recommended medium until 90% confluence.  Total RNA extraction was conducted using mirVana miRNA isolation kit with phenol (ThermoFisher, Waltham, MA), following supplier recommended protocols. Purified RNA was quantified using NanoDrop and Bioanalyzer was used to assess the quality of RNA. Samples with RNA integrity number >9 were used to construct libraries.  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] 10ng of input RNA from three replicates of each condition was synthesized into sequencing libraries using the Capture and Amplification by Tailing and Switching (Diagenode Inc., Denville, NJ). 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).
Experimental Factor: organism part
lung epithelium
Experimental Factor: strain
Nitrofen + AFSC-EVs