Exosome Isolation and RNA Extraction

Exosomes were isolated using the Exosome Precipitation Solution (EXOQ20A-1, SBI, Mountain View, CA) as previously mentioned (Wang et al., 2017 (link)). Briefly, 300 μL of plasma was thawed on ice and centrifuged at 3,000 g for 10 min to remove possible residual cell debris. After incubating with thromboplastin D (Thermo, Middletown, CA) at 37°C for 15 min and centrifuging, the 250 μL of supernatant was aspirated to a new tube and mixed with 65 μL of ExoQuick Solution. To digest free and lipoprotein-binding RNAs outside of small extracellular vesicles, RNaseA (Sigma, St. Louis, MO) was added to the mixture at a final concentration of 10 μg/mL. After keeping the mixture at 4°C overnight, murine RNase inhibitor (NEB, Ipswich, MA) was added at 150 units/mL before precipitation of exosomes by centrifuging at 1,500 g for 30 min. The exosome pellet was dissolved in 50 μL of phosphate-buffered saline (PBS) and subjected to RNA extraction immediately.