Physiological and morphological status of cells dictates RNA biosignature of their exosomes
P19 cells, a pluripotent embryonal cell line derived from mouse teratocarcinoma, can be stimulated with retinoic acid to induce neural differentiation forming P19 neurons (McBurney et al., 1982). P19 cells (undifferentiated cells abbreviated as UD-P19 cells) and P19 neurons were cultured in a defined medium lacking fetal bovine serum and conditioned medium was collected. Exosomes were purified from conditioned medium by ultracentrifugation method. Exosomes were characterized using a number of techniques according to the guidelines of ISEV. RNA was purified from exosomes using miRCURY™ RNA isolation kit (Exiqon, Inc.; now Qiagen) according to the instructions of the Supplier. Exosomal RNA concentration was measured using NanoDrop™ 8000 spectrophotometer. The approximate size of exosomal RNAs was analyzed on BioAnalyzer and by 5' end labeling and their separation on denaturing gel. Differential expression of RNAs in exosomes from P19 cells and P19 neurons was determined by RNA-Seq method. Double stranded barcoded cDNA libraries were generated and sequenced on Illumina NextSeq 500 platform. Three biological replicates were used from P19 cells. P19 neurons exosomes were from P19 neurons at 8, 10, 12 days of differentiation. RNA-Seq data was analyzed using bioinformatics pipeline. Overall design: Six biological replicates were analyzed by RNA-Seq method. Three samples were from undifferentiated P19 cells and three samples were from P19 neurons.