GSM7392779: EV miRNA, primary hepatocytes, HFHC diet, biol rep 3; Mus musculus; miRNA-Seq
The miRNA in primary hepatocytes derived EVs were extracted by SeraMir exosome RNA kit (SBI, USA). 1 μg total RNA for each sample was used for library construction. 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). The library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe).