GSM8339540: NZ-P3; Homo sapiens; miRNA-Seq
Total RNA was extracted from exosomes using the Total Exosome RNA & Protein Isolation Kit (Invitrogen, Cat. No: 4478545, USA). The "Isolate RNA–Purify the RNA" part was performed according to the manufacturer's instructions. Before the sequencing experiment, agarose electrophoresis was used to check the integrality of total RNA samples. Then the samples were quantified on the NanoDrop ND-1000 instrument (Thermo Fisher Scientific, USA). Library preparation for small RNAs was performed with NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, Cat. No: E7300, USA). The main procedures are as follows: total RNA samples were first pretreated as follows to remove some RNA modifications that interfere with small RNA-seq library construction: 3'-aminoacyl (charged) deacylation to 3'-OH for 3' adaptor ligation, 3'-cP (2', 3'-cyclic phosphate) removal to 3'-OH for 3' adaptor ligation, 5'-OH (hydroxyl group) phosphorylation to 5'-P for 5'-adaptor ligation, m1A and m3C demethylation for efficient reverse transcription. After the pretreatment, 3'-adapter ligation, 5'-adapter ligation, cDNA synthesis, and PCR amplification were sequentially carried out. Then, ∼134-160bp PCR amplified fragments (corresponding to ∼14-40nt small RNAs) were selected. The completed libraries were quantified by Agilent 2100 Bioanalyzer. Libraries were mixed in equal amounts according to the quantification results and used for sequencing on the instrument.