title: GSM8762003: Soft EV miRNA biol rep 3; Homo sapiens; miRNA-Seq
library_construction_protocol: EVs were isolated from culture media samples using an insulator-based dielectrophoresis approach. Total RNA was extracted from purified EVs using the miRNeasy Micro kit as per the manufacturer's suggested protocol. Size distribution and estimated RNA concentration were measured using the Agilent 6000 Pico Kit using Bioanalyzer. For library preparation, NEBNext Small RNA Sample Library Preparation kit was used with a modified approach for precise miRNA library size selection, which makes it possible for the kit to process low input (ng level) and low-quality (Bioanalyzer RIN value <3) RNA with better library recovery and miRNA reads alignment. Specifically, using ~8 ng total RNA as input, after 15 cycles of final PCR, libraries with unique indices were first equal-10 µl pooled, column cleaned up, and mixed with a custom-designed DNA ladder that contains 135 and 146 bp purified PCR amplicons. This size range corresponds to a miRNA library with 16-27 nt insert that covers all miRNAs. After high-resolution agarose gel electrophoresis and gel excision, the library pool ranging from 135 to 146 bp, including the DNA marker, was purified and quantified by NEBNext Library Quant kit (NEB) using QuantStudio 5 Real-Time PCR System. The first round of sequencing was performed on NextSeq 2000 sequencer to generate a few million reads to quantify the relative concentration of each library. The volume of each library was then adjusted to generate the expected number of equal reads [~4.5M read clusters] from each sample for final data analysis.