GSM4910000: AGS_shNC_1; Homo sapiens; miRNA-Seq
Gastric cancer cells were grown in 6-well plates and transfected with NC siRNAs and targeted siRNAs. After 48 hours, remove the medium and directly add 800μL Trizol into the 6-well plate to harvest samples. Total RNA was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. The RNA purity and concentration was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). Library was prepared with 1 μg total RNA for each sample. Total RNA was purified by electrophoretic separation on a 15% urea denaturing polyacrylamide gel electrophoresis (PAGE) gel and small RNA regions corresponding to the 18–30 nt bands in the marker lane (14-30 ssRNA Ladder Marker, TAKARA) were excised and recovered. Then the 18–30 nt small RNAs were ligated to a 5′-adaptor and a 3′-adaptor. The adapter-ligated small RNAs were subsequently transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen, USA) and then several rounds of PCR amplification with PCR Primer Cocktail and PCR Mix were performed to enrich the cDNA fragments. The PCR products were selected by agarose gel electrophoresis with target fragments 100~120 bp, and then purified by QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). After purification of microRNAs, the miRNA sequencing was performed according to the manufacturer's instructions.