EV-sorting small RNA in normal human plasma-EV compared with EV-depleted plasma

After mapping to transcriptome using bowtie2 and peak calling by RNA peak caller (cfPeak), count matrix was created by merge 3 pairs of samples. EV-sorting small RNA sites in normal human plasma total RNA-seq were annotated by comparing differentially expressed peaks (EV vs. EV-depleted Plasma). Peripheral whole blood samples were collected from individuals before therapy or surgery using EDTA-coated vacutainer tubes. Plasma was separated within 2 hr after collection by centrifuge at 1,900 g for 10 min at 4 ℃. All plasma samples were aliquoted and stored at -80°C. Plasma EV were purified by a membrane-affinity approach using exoRNeasy Midi Kit(Qiagen, 77144) following the manufacturer’s instructions. Plasma-nonEV (EV-depleted Plasma) component were collected from flow through part of MinElute spin column from kit. Both plasma EV RNA and Plasma nonEV RNA were extracted using QIAzol Lysis Reagent (QIAGEN, Beijing, China), and DNA contamination was removed by Recombinant DNase I (RNase-free) (TAKARA, Beijing, China). The total cfRNA libraries were prepared using our in-house protocol that improved from our previously published method, DETECTOR-seq. Library quantification was performed by Qubit dsDNA HS Kit and fragment size was checked by Agilent 2100 Bioanalyzer. Libraries were sequenced on DNBSEQ-T7 (MGI Tech.) with PE150.