small RNAseq of extracellular vesicles secreted by primary human airway epithelial cells

Characterization of the small RNA content of extracellular vesicles isolated from cell culture supernatants of primary human airway epithelial cells from 3 donors. Overall design: The sequencing of EV small RNA content was performed by System Biosciences. 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), 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) according to the manufacturer's instructions.