|Public on Mar 14, 2022
|gastric cancer cells SNU_cell_rep1
|gastric epithelial cells
|Homo sapiens; human gammaherpesvirus 4
|ebv status: positive
cell line: SNU-719
rna population: whole cell
|No special treatment other than growth protocol described above was conducted.
|SNU-719 (EBV positive gastric cancer cell line) and AGS (EBV negative gastric cancer cell line) were cultured in RPMI-1640 and F12, respectively. 5% fetal bovine serum without exosomes and penicillin and streptomycin (100U/ mL) were added into the culture medium before use. When the cells grow vigorously (with a degree of fusion up to about 70%), the supernatant is collected.
|Total RNA was extracted from cells and exosomes using the high pure RNA extraction kit (TRIzol) in accordance with the manufaxturer’s protocol. The OD260 and OD280 values and ratios of 1μL extracted samples were determined by ultraviolet spectrophotometer to determine the concentration and purity of extracted samples.
Approximately 1 μg of total RNA from each sample was used to prepare the miRNA sequencing library. The strips between 17 and 32 nt were cut by polyacrylamide gel electmphoresis（PAGE) and the products were recycled. T4 RNA ligase (Epicenter, San Diego, CA, USA) was used to connect the isolated small RNA to the 3' end and then the isolated small RNA to the 5 'end using the same method. The sRNA connected by the connector was used as the template for RT-PCR, and the single-stranded cDNA template was amplified into double-stranded cDNA, and then PCR amplification was conducted. Then the gel was cut and the target fragment library was recycled. The constructed library was used to detect quality and yield using Agilent 2100 Bioanalyzer and ABI StepOnePlus Real Time PCR System. The library with qualified quality test was sequenced.
|Illumina HiSeq 2500
|remove adapter, poly Ns, reads samller than 17 nts with fastqc
using miRDeep2, we align clean reads against sequences in miRBase22 library to predict known miRNA expression.
The expression levels were normalized to RPM (Reads per Million), that is number of reads mapping to miRNA/ number of reads in Clean data)×1000000
Differential expressed miRNAs were calculated using edgeR, and filtered using |log2(Fold Change)| >= 1 and p-value < 0.05
Assembly: miRBase22(reads were blasted directly against library)
Supplementary files format and content: comma seprated values; expressions of human miRNAs in RPM
Supplementary files format and content: comma seprated values; expressions of EBV miRNAs in RPM
|Mar 10, 2022
|Last update date
|Mar 15, 2022
|The First Affiliated Hospital of Fujian Medical University
|Department of Pathology
|20 Cha Zhong Lu
|Study on the Expression of miRNA in Exosome of EBV-positive Gastric Carcinoma Cells