GSM5543553: Sample_013; Homo sapiens; miRNA-Seq
Blood samples (10ml) were collected in EDTA collection tubes and plasma was isolated  within next 2 hours by centrifugation at 2000 Xg for 20 min. Plasma was stored at -80oC until  analysis. Total RNA, including miRNA was isolated from 1 ml frozen plasma samples using  exoRNeasy Serum/Plasma Maxi Kit (Qiagen, Valencia, CA, USA) [31]. Briefly, pre-filtered  plasma (0.8 μm syringe filter) was mixed 1:1 with 2x binding buffer (XBP) and added to the  exoEasy membrane affinity column to bind the EVs to the membrane. After centrifugation, the  flow-through was discarded, and wash buffer (XWP) was added to the column to wash off nonspecifically  retained material. After another centrifugation and discarding of the flow-through,  the vesicles were lysed by adding QIAzol to the spin column, and the lysate was collected by  centrifugation. The miRNeasy Serum/Plasma Spike-In Control (Qiagen, Valencia, CA, USA)  was added. Following addition of chloroform, thorough mixing, and centrifugation to separate  organic and aqueous phases, the aqueous phase was recovered and mixed with ethanol. The  sample-ethanol mixture was added to Rneasy MinElute spin column and centrifuged. The  column was washed once with buffer RWT, and then twice with buffer RPE followed by elution  of RNA with water. The purity and concentrations of total RNA of the plasma samples were  measured with NanoDrop ND-1000. 3' and 5' adapters are ligated to mature miRNAs. The ligated miRNAs are then  reverse transcribed to cDNA using a reverse transcription (RT) primer with unique molecular  indices (UMI). After library amplification, a cleanup of the miRNA library is performed using a  streamlined magnetic bead-based method and quality control (QC).