GSM5942975: A013  after acupuncture; Homo sapiens; miRNA-Seq
Exosome isolation was performed with an exoEasy Maxi kit (product no. 76064; Qiagen, Hilden, Germany) according to the manufacturer's protocol with minor modifications. Briefly, the platelet-free plasma was filtered with a 0.8-μm pore filter (product no. SLAA033SB; Millipore, Burlington, Massachusetts, United States) to exclude large particles. An equivalent volume of XBP buffer was added to 500 μL of platelet-free plasma, and the sample was mixed well by gently inverting the tube five times. The mixture was added to the exoEasy spin column and centrifuged at 500 g for 1 min at room temperature, and the flow-through was discarded. Next, 10 mL of buffer XWP was added, and the column was centrifuged at 5,000 g for 5 min to remove residual buffer from the column. The column was transferred to a fresh collection tube, and 400 μL of XE buffer was added to the column membrane, which was then incubated for 1 min. The column was centrifuged at 500 g for 5 min to collect the eluate. The methods for identifying the exosomes mainly included transmission electron microscopy (TEM; FEI Tecnai Spirit T12; FEI, Hillsboro, Oregon, United States), exosome size analysis (NanoFCM; Xiamen, China), and western blotting. Total RNA was extracted from exosomes with a miRNeasy Serum/Plasma kit (product no. 217184; Qiagen). RNA purity was checked using a NanoPhotometer spectrophotometer (Implen, München, Germany). RNA concentrations were measured with a Qubit RNA Assay Kit in a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, California, United States), and RNA integrity was assessed with the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, California, United States) to ensure that the samples were suitable for sequencing. RNA libraries were prepared for sequencing using standard Illumina protocols