GSM9372171: e-miRNA_plasma_ICAD_91; Homo sapiens; miRNA-Seq
Exosome extraction and purification: Plasma samples were centrifuged at 1500 rpm for 5 min for debris removal. For exosome isolation, a 1.5 ml Eppendorf tube was filled with supernatant. This was later combined with ExoQuick (System Biosciences, Palo Alto, CA) at a 1:4 ratio, incubated (30 min, 4 °C), and centrifuged for an additional 30 min.  Exosome total RNA isolation: The SeraMir Exosome RNA Purification Column kit (Cat #RA808A-1, System Biosciences, Palo Alto, CA) was used to isolate total RNA. Concentration of small RNA was measured with the Small RNA kit for Bioanalyzer 2100 Bioanalyzer Systems (Agilent, Santa Clara, CA). A concentration of 200 pg/ul small RNA was used for subsequent library generation. The NGS library was generated using the CleanTag Small RNA Library Preparation Kit (TriLink, Cat# L-3206) and quantified using the High Sensitivity DNA Reagents (Agilent, Santa Clara, CA, PO# G2933-85004) and High Sensitivity DNA Chips (Agilent, Santa Clara, CA, PO# 5067–4626). NGS libraries were pooled using the TBE gel (Invitrogen by Life Technologies, Grand Island, NY, Ref# EC6215) and quantified using the High Sensitivity DNA 1000 Screen Tape (Agilent Technologies, Santa Clara, CA, PO # 5067–5584), High Sensitivity D1000 reagents (, Agilent Technologies, Santa Clara, CA, PO# 5067–5585), 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 (Illumina, San Diego, CA, Cat #FC-404-2005).