title: GSM6792006: plasma-derived HC-exosomes Nymber 5; Homo sapiens; RNA-Seq
library_construction_protocol: RNA extraction from exosomes  The entire method for isolating RNA from exosomes with exoRNeasy Midi Kit (Qiagen) consists of two steps: exosome purification and RNA isolation. Isolating RNA from exosomes consisted of two steps: exosome purification and RNA isolation. For exosome purification, the plasma sample was mixed with buffer XBP (1:1) and centrifuged for 1 min at 500 x g to bind to an exoEasy membrane affinity spin column. The bound exosomes were washed with 3.5 ml buffer XWP after 1 min of centrifugation at 500g, and then lysed by adding 700 l QIAzol after 5 min of centrifugation at 5000g.  In the RNA extraction stage, 90 µl chloroform was added to the QIAzol eluate, which was then shaken for 15 seconds. The aqueous phase was then collected and combined with 2 volumes of 100% ethanol. Total RNA, including miRNA, attached to the spin column after 15 seconds of centrifugation at 8000 x g, was washed three times and eluted in 14 lµ RNase-free water.. All protocols were provided by Qiagen. The enzyme Bacteroides I (New England Biolabs, P0735S) and RNase Inhibitor (Enzymatic, Y9240L) were utilized after RNA extraction following the manufacturer's protocol to eliminate residual heparin potentially transferred from the heparin blood collection tubes. After incubation for 1 hr at 30C and 1 min at 99.9C, the RNA was stored at -80°C and then used as RNA templates to produce a cDNA library for sequencing. MiRNA libraries were prepared using Qiagen's QIAseq miRNA Library Kit with 5 μl of total RNA from each sample. This was followed by completing universal cDNA synthesis with UMI assignment, cDNA cleaning, library amplification, and library purification. The resulting libraries were quantified using the Denovix hsDNA assay, and quality was assayed using a hsDNA chip on an Agilent Bioanalyzer.