GSM6857530: FF-EV_20; Homo sapiens; miRNA-Seq
Approximately 1 mL of each FF sample was centrifuged at 10,000 g for 40 min at 4 ℃ in the Kubota Model 7000 ultra-centrifuge (KUBOTA Co., Tokyo, Japan). The supernatant was filtered using a 0.22 µm filter (Millex-GV 33 mm, Millipore) and then ultracentrifuged at 110,000 g for 70 min at 4 ℃ using an MLS50 rotor (Beckman Coulter Inc., USA). The pellet was washed with PBS, ultracentrifuged with the same conditions, and resuspended in PBS to extract small EVs. Then, total RNAs were extracted using the miRNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). Small RNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA). Then, PCR products were separated by electrophoresis (120 V, 60 min), and DNA fragments corresponding to 140–160 bp (the length of small non-coding RNA plus the adaptors) were recovered.