GSM5322566: WT_25J_ctrl_EV; Homo sapiens; ncRNA-Seq
EVs were harvested from 15 ml cell culture supernatants using the ExoQuick-TC EV isolation kit. Total EV RNA was isolated with the SeraMir Exosome RNA Purification Column kit (System Biosciences, Cat. #RA808A-1) according to the manufacturer's instructions. For each sample, 1 µl of the final RNA eluate was used for measurement of small RNA concentration by Agilent Bioanalyzer Small RNA Assay using the Bioanalyzer 2100 Expert instrument (Agilent Technologies, Santa Clara, CA, Cat. # 5067-1548). Small RNA libraries were constructed with the CleanTag Small RNA Library Preparation Kit (TriLink Biotechnologies, San Diego, CA, Cat. # L-3206) according to the manufacturer's protocol. The final purified library was quantified with High Sensitivity DNA Reagents (Agilent Technologies, PO # G2933-85004) and High Sensitivity DNA Chips (Agilent Technologies, PO # 5067-4626). The libraries were pooled, and the 140 bp to 300 bp region was size selected on an 8% TBE gel (Thermo Fisher Scientific, Ref. # EC6215). The size selected library was quantified with High Sensitivity DNA 1000 Screen Tape (Agilent Technologies, PO # 5067-5584), High Sensitivity D1000 reagents (Agilent Technologies, PO # 5067-5585), and the TailorMix HT1 qPCR assay (SeqMatic, Fremont, CA, Cat. # TM-505). Libraries were sequenced with Illumina 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) according to the manufacturer's instructions.