Isolation and Characterization of CSF sEVs

sEVs were isolated from CSF by differential centrifugation, according to the previous publications [35 (link)]. After removing cells and other debris by centrifugation at 2000 × g for 30 min, the supernatant was centrifuged at 12,000 × g for 45 min to remove shedding vesicles and the other vesicles with bigger sizes. The supernatant was centrifuged at 110,000 × g for 70 min and resuspended in 10 mL PBS. Finally, the suspension was recentrifuged at 110,000 × g for 70 min (all steps were performed at 4 °C); sEV were collected and resuspended in 50 μL PBS for further trans-omics RNA sequencing. The transmission electron microscopy assay of CSF sEV pellet was examined and photographed with an FEI Tecnai spirit TEM T12(FEI Tecnai Spirit 120 kv), according to the previous publications [35 (link)].