Exosomal RNA Extraction and Sequencing

As reported previously [11 (link)], RNA extraction was performed using the Total Exosome RNA and Protein Isolation Kit (catalog # 4478545; Invitrogen, USA) according to the provided instructions. 200 ng-1 μg RNA in final volume of 30 μL solution was collected for each sample. Total RNA quantity and quality (260/280 absorbance ratio) were assessed using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer to test concentration and inorganic ions or polycarbonate contamination. miRNA sequence was isolated by BGI Company (China) based on previous instructions [12 (link)]. cDNA libraries were constructed using the Ion Total RNA-Seqv2 kit (Life Technologies, USA) (n = 3 for each group) and purified using AMPure beads (Beckman Coulter). Emulsion PCR and enrichment of cDNA-conjugated particles were performed with an Ion OneTouch 200 Template Kit v2 DL (Life Technologies). The final cDNA samples were sequenced single end on the HiSeq 2000 System with a 50 bp read length.