title: GSM7865817: D2; Ceratotherium simum; miRNA-Seq
library_construction_protocol: EVs were isolated using the dual-ended approach of ultracentrifugation followed by precipitation and elution through size exclusion chromatography columns. Initially, in three technical replicates, frozen-thawed collections containing 2 mL of follicular fluid aspirate from individual animals were subjected to an initial ultracentrifugation using an SW 55Ti rotor on the Optima XE-90 Ultracentrifuge (Beckman Coulter; Pasadena, CA, USA.) system at 120,000 xg for 70 min at 4°C, Pelletized EVs were then washed using 3 mL of Dulbecco's Phosphate Buffered Saline without calcium and magnesium chloride (DPBS (1X)) (Sigma-Aldrich; St. Louis, MO, USA.) and ultracentrifuged once more under the same parameters. Following ultracentrifugation, washed EV pellets were resuspended in Exo-spin™ Buffer and left to precipitate overnight at 4°C prior to being processed through Exo-spin™ mini columns (CELL guidance systems; St. Louis, MO, USA.) according to the manufacturer's protocol. Ensuing 180 µL EV aliquots were then stored at -80°C for RNA extraction. 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.