title: GSM9253793: extracellular vesicles, postnatal day 7 mouse testis EVs (PND7) treated, biol rep 2; Mus musculus; miRNA-Seq
library_construction_protocol: Each preparation of PND7 testis EVs was isolated from ten PND7 C57/BL6 male mice. The tunica albuginea was removed using forceps to release the seminiferous tubules. The decapsulated testes were digested by 1ml Accumax (Thermo) per 20 mg tissue at room temperature for 1 hour. Tissues were gently pipetted up and down 20 times to maximize the cell yield followed by filtering through a 40 μM strainer. The digestion was stopped by the addition of 1 volume of PBS and the cells were pelleted by centrifugation at 300g 10 min. The supernatant of the single-cell suspension was collected and the EVs were isolated using exoEasy Maxi Kit (Qiagen, 76064) according to the manufacturer's instructions. The EV preparation was further concentrated by ultracentrifugation (Hitachi CS-150GXII Micro UITRAcentrifuge) at 100,000g for 90 min at 4°C. The EV pellet was resuspended in 20 μl PBS and stored at -80℃ for further analysis. treat EVs with proteinase K, PMSF and RNase A.RNA was extracted using miRNeasy Mini Kit.