title: GSM5392917: ERSIAS0004; Homo sapiens; miRNA-Seq
library_construction_protocol: Plasma sample with the volume of 500 μl was centrifuged at 1500 rpm for 5 min to remove residual cells and debris. The supernatant was transferred to a new 1.5 ml Eppendorf tube for exosome isolation. ExoQuick (Cat# EXOQ5A-1, System Biosciences, Palo Alto, CA) was added to the supernatant at 1:4 ratio (ExoQuick:Supernatant), mixed gently, and allowed to incubate for 30 min at 4 °C. After the incubation, the admixture was centrifuged at 1500 rpm for additional 30 min to recover the exosomes. Using ExoQuick-based precipitation techniques for exosome isolation and recovery is broadly recognized for better specific exosomal miRNAs recovery and lower intra-assay coefficients of variability (CVs) compared to other commercial exosome isolation kits, and is also backed in nearly 600 peer-review citations. Exosomes extracted from the samples were processed for total RNA isola- tion using the SeraMir Exosome RNA Purification Column kit (Cat #RA808A-1, System Biosciences, Palo Alto, CA) according to the manufacturer's instructions. For each sample, 1 μl of the final RNA eluate was used for meas- urement of small RNA concentration by Agilent Bioanalyzer Small RNA Assay using Bioanalyzer 2100 Expert instrument (Agilent Technologies, Santa Clara, CA). A small RNA concentration of 200 pg/ul was considers as a cutoff for subsequent library preparation. Small RNA libraries were prepared for sequencing using standard Illumina protocols. Small Small RNA libraries were constructed with the CleanTag Small RNA Library Preparation Kit (Cat# L-3206, TriLink BioTechnologies, San Diego, CA) according to the manufacturer's protocol The final purified library was quanti- fied with High Sensitivity DNA Reagents (PO# G2933-85004, Agilent Technologies, Santa Clara, CA) and High Sensitivity DNA Chips (PO# 5067–4626, Agilent Technologies, Santa Clara, CA). The libraries were pooled, and the 140 bp to 300 bp region was size selected on an 8% TBE gel (Ref# EC6215, Invitrogen by Life Technologies, Grand Island, NY). The size selected library was quantified with High Sensitivity DNA 1000 Screen Tape (PO #5067–5584, Agilent Technologies, Santa Clara, CA), High Sensitivity D1000 reagents (PO# 5067–5585, Agilent  Technologies, Santa Clara, CA), and the TailorMix HT1 qPCR assay (Cat# TM-505, SeqMatic, Union City, CA),  followed by a NextSeq High Output single-end sequencing run at SR75 using NextSeq 500/550 High Output v2  kit (Cat #FC-404-2005, Illumina, San Diego, CA) according to the manufacturer's instructions. Then, the open-source tools including FastqMcf, part  of the EA-utils package, and PRINSEQ were used to detect and remove N's at the ends of reads, trim sequencing  adapters, and filter reads for quality and length. The improved set of sequence reads were mapped to the  reference genome using Bowtie and expression levels for exosomal miRNAs were calculated using R statistical  environment