Instrument: HiSeq X Ten
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Isolation of exosomes using ExoQuick（SBI,Palo Alto,CA,US) according to the manufacturer's instructions.Exosomal RNA was isolated from samples using RNAiso Plus Total RNA extraction reagent (TAKARA, Dalian, China) according to the manufacturer's instructions and the RIN number was checked to detect RNA integrity by Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Further purification of qualified total RNA using RNAClean XP Kit (Beckman Coulter, Inc. Kraemer Boulevard Brea, CA, USA) and RNase-Free DNase Set(QIAGEN, GmBH, Germany). The total RNA were treated with the RiboZero rRNA Removal Kit (Epicentre, WI, USA) to delete rRNA and digested linear RNA using RNase-R (Epicentre, WI, USA) according to the manufacturer's instructions. Next, strand-specific libraries were constructed by using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme, Nanjing, CHN) following the manufacturer's instructions. In brief, The enriched circRNAs were fragmented using fragmentation buffer and then reverse transcribed into first-strand cDNA and second-strand cDNA in orderly. Then, the cDNA fragments were purified, subjected to end-repair, and modified to add poly (A) to the 3' end. The sequencing adapters were ligated to the cDNA fragments. The double strand cDNA are digested by uracil-DNA glycosylase (UDG) (Cwbio, Beijing, CHN) to remove the second-strand cDNA before sequencing. The purified ligation products were performed 13-15 cycles of PCR amplification. The PCR amplification products of cDNA were purified with Agencourt AMPure XP Beads(Beckman Coulter, Inc. Kraemer Boulevard Brea, CA, USA) and then sequenced by Illumina Hiseq X Ten system (Illumina, San Diego, CA, USA) on paired-end(PE) mode with length 150 bases following the vendor's recommended protocol.