title: GSM8651726: Plasma exosome non-OSA_4; Homo sapiens; miRNA-Seq
library_construction_protocol: Peripheral blood samples from individuals were collected in EDTA tubes following a regular venipuncture procedure. After centrifugation at 3,000 ×g for 15 min at 4 °C, the plasma was aspirated and stored at −80 °C before use. The UC method was optimized according to the method previously described (Théry C. et al. 2006).  After thawing at 37 °C, 1 mL plasma samples were centrifugated at 3,000 ×g for 15 min to remove cell debris. Repeat the above steps. Then, the supernatant was diluted by five-fold volume of PBS, centrifuged at 10,000 ×g for 20 min, and processed through a 0.22 μm filter to remove large particles. The supernatant was ultracentrifuged at 100,000 ×g, 4 °C for 60 min to pellet the exosomes. The pellet was resuspended in phosphate-buffered saline (PBS) and centrifuged again at 100,000 ×g 4 °C for 4 h. After PBS washing, the exosomes pellet was re-suspended in 100 µl PBS. Total RNA was extracted and purified from plasma exosome using miRNeasy Serum/Plasma Advanced Kit (Qiagen, cat. No. 217204) according to the kit instruction. RNA concentration and purity were evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA). A total amount of 250pg—10ng RNA per sample was used as input material for sequencing libraries using the SMARTer Stranded Total RNA-Seq Kit V2 (Takara Bio USA, Inc.) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. For small RNA libraries, a total amount of 1ng-500ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using QIAseq miRNA Library Kit (Qiagen, Frederick, MD) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Reverse transcription (RT) primers with unique molecular indices (UMIs) were introduced to analyze the quantification of miRNA expressions during cDNA synthesis and PCR amplification. At last, library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR. The clustering of the index-coded samples was performed on acBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.