Illumina HiSeq 2000 paired end sequencing; RNA-seq of non coding RNA from serum exosomes of GC patients and healthy individuals
RNA-seq of non coding RNA from serum exosomes of GC patients and healthy individuals
Serum samples from 7 GC patients and 2 healthy controls (HC) who had no history of basic or chronic diseases, and 36 tumor tissues and matched adjacent non-tumor tissues from GC patients who did not receive radiotherapy and chemotherapy before surgery were collected from the First Affiliated Hospital of Jinan University. Samples were stored at −80°C until use. Exosomes were first isolated from serum using the ExoQuick™ (SBI, USA) according to the manufacturer's instructions. Briefly, 300 μl of cell-free serum samples were moved into a tube and mixed with 75 μl of ExoQuick reagent. Next, the mixture was incubated at 4°C overnight and then centrifuged at 1500 g for 30 min. Then, the pelleted exosomes were re-suspended in 40 μl PBS. Finally, RNA of exosomes was extracted with the miRNeasy micro kit (Qiagen, USA) based on the manufacturer's instructions, and was subjected to next step. The clustering of the index-coded samples was first carried out on a cBot Cluster Generation System using TruSeq SR Cluster kit v3-cBot-HS (Illumina). Then, the libraries were sequenced and 50 bp single-end reads were generated. Next, clean data were acquired by eliminating the low quality reads and adaptors in HTseq-count (version 0.7.2), FastQC (version 0.11.7) and Trimmomatic (version 0.38) software.