GSM5741661: T4; Homo sapiens; ncRNA-Seq
Serum isolated from coagulation‐promoting vacuum tubes within 2 hours was immediately transferred to a 1.5 mL Eppendorf tube and immediately centrifuged at 1500 g for 5 minutes and then 13800 g for 5 minutes at 4°C to eliminate cell sediments. ExoQuick™ solution (EXOQ5A‐1; SBI System Biosciences, USA) was mixed evenly with supernatant according to the manufacturer's instructions. Subsequently, incubate at 4°C for 30 minutes, followed by centrifuged twice at 4°C (1500g, 30 minutes and 1500g, 5 minutes), supernatants were discarded, and the exosome pellets were resuspended in 250 μL PBS and stored at −80°C for further analysis. Total exosomal RNA was extracted from miRNeasyMicro Kit (Qiagen) according to the manufacturer's instructions. Briefly, rRNAs were removed from total RNA using Ribo-Zero rRNA Removal Kits (Illumina, USA) according to the manufacturer's instructions. Subsequently, rRNA-depleted RNAs were used to constructed RNA libraries with TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA). BioAnalyzer 2100 system (Agilent Technologies, USA) was performed to control the quality and quantified. Libraries mentioned above were denatured as single-stranded DNA molecules, after captured on Illumina flow cells, amplified as clusters and finally sequenced on Illumina HiSeq Sequencer for 150 cycles.