Extraction and Analysis of Extracellular Vesicle RNA

Total RNA was extracted using the Trizol™ method following the manufacturer's protocol (31 (link), 32 (link)). Briefly, Trizol™ reagent (Thermo Fisher Scientific) was admixed to either whole cell suspension, APs, MVs or EXs (9 part Trizol: 1 part cells/vesicles). The Trizol-vesicle solution was triturated or vortexed to ensure vesicle lysis prior to addition of MgCl₂ solution (Sigma) and chloroform. Each mixture was vortexed vigorously, incubated at room temperature (RT) for and centrifuged prior to transfer of the aqueous phase to fresh 2 mL micro tubes. Then, isopropanol was added and the samples were repeatedly inverted and incubated at RT. The tubes were then incubated at −20°C for 1 h (or overnight). Following incubation, the samples were centrifuged at 12,000 × g for 10 min at 4°C to collect RNA pellet. The RNA pellets were washed in RNAse-free 75% ethanol twice prior to removal of the supernatants and allowing the RNA pellets to air dry. Finally, the RNA was resuspended in 10–20 μL RNase-free water (Invitrogen; depending on the size of the RNA pellets).
Vesicle-derived RNA was quantified and evaluated for quality (as described in the Supplementary Material). RNA samples of sufficient quality were initially subjected to qRT-PCR to confirm the presence of miRNAs prior to subjecting RNA to deep sequencing using the Illumina® NextSeq500 system (below).