GSM5627647: 3R; Homo sapiens; miRNA-Seq
The filtrated conditioned medium stored at -80°C was thawed and ultracentrifuged at 110,000 g, 4°C for 2.5 hrs to obtain EV pellet using Sorvall Discovery 90SE centrifuge (Hitachi) with AH-629 swinging bucket rotor (ThermoFisher Scientific). The EV pellet was washed with PBS at the same speed for 0.5 hrs. RNA was isolated using mirVana miRNA Isolation Kit (ThermoFisher Scientific) following the manufacturer's instruction. Specifically, EV pellets isolated from the ultracentrifugation were disrupted with the Lysis Solution. Following organic extraction using acid-phenol: chloroform, total RNA was isolated and eluted in nuclease-free water. RNA quantity and quality isolated from EVs were evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies). SmRNA-sequencing libraries were prepared from 1.2 to 10 ng of total RNA using the QIAseq miRNA Library Kit (Qiagen, Germantown, MD) as per the manufacturer's instructions. This system offers a built-in Unique Molecular Identifier (UMI) application, which is used to eliminate possible PCR duplicates in sequencing datasets and therefore facilitate unbiased gene expression profiling. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled libraries were diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol. The denatured libraries were loaded onto an SP flow cell on an Illumina NovaSeq 6000 and run for 71 cycles using a single-read recipe according to the manufacturer's instructions.