title: GSM6855679: SUM_2; Bos taurus; miRNA-Seq
library_construction_protocol: For EV isolation, follicular fluid samples were subjected to a series of centrifugations starting at 500 xg for 10 minutes to remove cells, followed by 3000 xg for 10 minutes to remove the cellular debris, and at 12,000 xg for 30 min to remove protein aggregates and large particles. All centrifugation steps were performed at 4 °C. The supernatant of FF samples was filtered through a 0.22 µM sterile filter to remove particles larger than 200 nm. 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 OC using the Beckman SWTi55 rotor. The EVs pellet was washed with sterile PBS and then centrifuged again at 120,000 xg for 70 min. Finally, EVs were resuspended in 500 µL of PBS and stored at −80 °C until further 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. 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.