GSM9629634: Sample 3_2A; Ceratotherium simum simum; ncRNA-Seq
For the GC ampless, total RNA, including miRNA, was isolated using the Qiagen miRNeasy Micro Kit (Qiagen, Germantown, MD) per the manufacturer's instructions. Briefly, granulosa cells were incubated with Qiazol lysis reagent and vortexed to homogenize the cells. After 5 min, chloroform was added to the sample and centrifuged at 10,000 x g for 15 min to separate RNA and protein. The RNA phase was added to an elution column and eluted in water after multiple washes. For the follicular fluid (FF) samples, FF was centrifuged at 300 x g for 10 min, the supernatant was re-centrifuged at 2000 x g for 10 min, and again at 16,500 x g for 30 min. The final supernatant was passed through a 0.20-μm sterile syringe filter, removing particles larger than 200 nm in diameter. For the EV isolation, 2 ml of pre-centrifuged follicular samples were subjected to an ultracentrifugation procedure at 120,000 xg for 70 min at 4 C using the Beckman SWTi55 rotor. The EVs pellet was washed with sterile PBS and then centrifuged again at 120,000 xg for 70 min. The washed pellets were then resuspended in DPBS.  To precipitate EVs,  Exo-spin™ buffer was added to each sample and incubated overnight at 4°C. The precipitated EVs were purified using Exo-spin™ mini columns according to the manufacturer's instructions. Final 180 µL EV aliquots were stored at −80°C for subsequent characterization and analysis. Total RNA including miRNAs was isolated from EVs using a Norgen Exosomal RNA Isolation kit (Norgen, Canada), according to the manufacturer's instructions. On-column DNA digestion was performed to remove genomic DNA contaminants. For GC samples library preparation kit was SMARTer smRNA-Seq Library Preparation Kit (Takara Bio) per manufacturer's instructions. For FF-EV samples, Small-RNA libraries were prepared for next-generation sequencing (NGS) using a TruSeq Small RNA Library Prep Kit (Illumina) according to the manufacturer's instructions.