GSM5834938: AI-N3; Homo sapiens; RNA-Seq
extracellular vesicles RNA were isolated by affinity-based binding to spin columns using an exoRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany). RNA libraries were prepared for sequencing using SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian. The RNA was then fragmented chemically into about 100 nucleotides in length using fragmentation buffer (Illumina, Inc.). The RNA was precipitated using the aqueous phase by mixing with isopropyl alcohol. The samples were incubated at 15ºC to 30℃ for 10 minutes and centrifuged at 12,000 x g for 10 minutes at 4℃. The ribosomal RNA-depleted RNAs were then used to construct RNA libraries with TruSeq Stranded Total RNA Library Prep kit (Illumina, CA, USA). The strand-specific library construction uses dUTP instead of dTTP to label the second-strand cDNA during synthesis. Uracil-DNA glycosylase (UDG) was used to hydrolyze the strand before polymerase chain reaction (PCR) enrichment to ensure that the final sequencing data was from the first cDNA strand.