GSM9306831: subject5_Urine_month3_rep2; Homo sapiens; miRNA-Seq
Approximately 3.5 mL of urine was centrifuged at 2,000 × g for 30 minutes at 4°C to remove cells and debris. From the resulting supernatant, 3 mL was mixed with an equal volume of Total Exosome Isolation Reagent for urine (Thermo Fisher Scientific, Waltham, MA) and incubated at room temperature on a shaker at 100 rpm for 1 hour. The mixture was then centrifuged at 3,000 × g for 1 hour at 4°C to precipitate the EVs. The resulting EV pellet was resuspended in 250 μL of phosphate-buffered saline and subjected to RNA extraction using the MagMAX mirVana Total RNA Isolation Kit with the KingFisher Apex automated system (Thermo Fisher Scientific), following the manufacturer's instructions. RNA was initially eluted in 30 μL of elution buffer. The eluate was then concentrated at 60°C for 30 to 45 minutes using a centrifugal concentrator (Eppendorf, Hamburg, Germany) and finally resuspended in 5 μL of nuclease-free water. Purified RNA samples were stored at −80°C until further analysis. Small RNA libraries were prepared using 5 μL of total RNA and the QIAseq miRNA Library Kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions. Library concentrations were quantified using the Qubit Flex Fluorometer (Thermo Fisher Scientific) with the Qubit™ dsDNA High Sensitivity Assay Kit. Prepared libraries were stored at −20°C until sequencing. Sequencing was performed on a NextSeq 550 system (Illumina, San Diego, CA) using 2 pmol of denatured libraries and single-end 75 bp reads, following the manufacturer's protocol.