GSM6909093: EV_B_42; Bos taurus; miRNA-Seq
For EV isolation, oviductal organoid conditioned media 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 the 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 °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. EVs were resuspended in 500 µL of PBS and further standardized by elution in 180 µL of sterile DPBS (1X) (ThermoFisher Scientific 14190-144) using the Exo-spin mini columns (CELL guidance systems EX03-50) according to the manufacturer's protocol. Isolated EVs were stored at -80°C for further 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.