GSM7692519: 22082R-05-13; Mus musculus; miRNA-Seq
Total RNA was extracted from BV2 cells and BV2 cell-derived extracellular vesicles using the Qiagen miRNeasy Mini Kit (Cat. No. 217004). 20 µL of EV fraction was resuspended in 700 µL of trizol lysis buffer. BV2 cell pellets were harvested in 700 μL of trizol lysis buffer. Added 140ul of chloroform was added to each sample and shook vigorously. Samples were then centrifuged for 15 min at 12,000 x g at 4°C. After centrifugation, the sample separated into 3 phases: an upper, colorless, aqueous phase containing RNA; a white interphase; and a lower, red, organic phase. The top aqueous phase was carefully isolated without disturbing the other phases. The top aqueous phase was transferred to a new collection tube and 1.5 volumes of 100% ethanol was added and mixed by pipetting up and down. 700 μL of the sample was added to the provided spin column in collection tubes. Samples were spin at ≥8000 x g for 15 s at room temperature and the flow-through was discarded. This was repeated for the rest of the sample. Bound RNA was washed 2 times using the included wash solution (Buffer RPE). Finally, a preheated elution solution (RNase-free water) was used to elute the RNA in 40 uL volume for cells and 20 uL for extracellular vesicles. cDNA Libraries were prepared for small RNAs using the SMARTer smRNA-seq kit. A total of 18 cycles of PCR were carried out to obtain a good yield of cDNA from cells and EVs. Final library quality was verified with Qbit and bioanalyzer. Negative (no RNA) and positive controls provided expected results. Next-generation RNA sequencing was performed using a HiSeq X Illumina 2x150, 40M total reads per sample (20M each direction) at Admera Health.