B004 before sham acupuncture, biological replicate 1
We used the exoEasy Maxi Kit (product no.: 76064, Qiagen, Hilden, Germany) to isolate and purify the exosomes from serum samples of the MWoA patients, according to the manufacturers protocols with minor modifications. We used transmission electron microscopy (TEM; FEI Tecnai Spirit T12; FEI, Hillsboro, Oregon, United States), exosome size analysis (NanoFCM; Xiamen, China) to visualise and analyse the size range of serum exosomes of MWoA patient, respectively. Exosomal markers like CD9 and CD63 were characterised by western blotting (WB). Total RNA was extracted from exosomes using the miRNeasy Serum/Plasma kit (product no. 217184; Qiagen) according to the manufacturers protocols. RNA purity and concentrations were evaluated by the NanoPhotometer spectrophotometer (Implen, Mnchen, Germany) and the Qubit 2.0 fluorometer (Life Technologies, Carlsbad, California, United States), respectively. The RNA integrity was assessed with the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, California, United States). Then, RNA samples (2.5 ng) were used for small RNA sequencing library construction with the NEB Next Ultra small RNA Sample Library Prep Kit (New England Biolabs, Ipswich, Massachusetts, United States) and the libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, California, United States).