title: NextSeq 550 sequencing; Rapid purification and multiparametric characterization of circulating small extracellular vesicles utilizing a label-free lab-on-a-chip device
design: Rapid purification and multiparametric characterization of circulating small extracellular vesicles utilizing a label-free lab-on-a-chip device
library_construction_protocol: One mL aliquots of archival serum (n = 6), plasma (n = 2), and urine (n = 3 with a pair of duplicates) from patients with genitourinary malignancies (bladder, kidney and prostate cancers), were obtained from the University of Cincinnati Biorepository. Basic demographic and clinical characteristics were provided for each patient. Exosomes were simultaneously extracted from various biofluids for downstream analysis via flow cytometry and miRNA-seq using the iDEP device. Total RNA extraction from exosomes was performed using the miRNeasy Micro kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. miRNA library preparation and sequencing was performed by the Genomics, Epigenomics and Sequencing Core at the University of Cincinnati College of Medicine. Library preparation was performed using the NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, Ipswich, MA) as per the manufacturer's protocol with modification for precise miRNA library size selection to increase small RNA detection sensitivity and specificity. After 15 cycles of PCR for indexing and library enrichment, an equal volume of 10 μL PCR mix was pulled from each library, followed by column clean up. Pooled samples were mixed with 135 bp and 146 bp custom-designed DNA ladder corresponding to miRNA library with 16-27 nt. Precise size selection of 135-146 library was performed using agarose gel electrophoresis and quantified by NEB Next Library Quant kit (NEB) using Quant Studio 5 real-time PCR system (Thermo Fisher, Waltham, MA). An initial round of sequencing was performed to quantify the relative concentration of each library by generating a few million reads using a NextSeq550 sequencer (Illumina, San Diego, CA).