GSM8986450: oviductal_fluid_1; Oryctolagus cuniculus; miRNA-Seq
After at least 14-15 hours, all animals were euthanized (with barbituric overdose previous sedation), and after a laparotomy, ovaries and the rest of the reproductive tract were extracted. The presence and number of corpus luteum on ovarian surface were determined to confirm that all females had ovulated, and a mean of 11.8±2.3 corpus luteum/doe was observed. Both oviducts and uteri from female rabbits were transported to the laboratory on ice. The oviducts and uteri were carefully trimmed to remove associated tissues and washed in cold Ca2+ and Mg2+- free phosphate-buffered saline (PBS−). Each oviduct was flushed (1 mL cold PBS−) from ampulla to isthmus, while each uterus was flushed (2.2 mL cold PBS−) towards the uterine tubal junction. All manipulations were performed at 4 °C. To remove cells and cellular debris, each oviduct and uterine flush sample was centrifuged once at 300 × g for 7 min. Subsequently, the supernatant was centrifuged again at 10,000 × g for 30 minutes. The resulting supernatants were individually passed through a 0.22-μm filter and stored at 4°C until the isolation of EVs. Subsequently, the method of  Cañón-Beltrán et al. [https://doi.org/10.1007/978-1-0716-1246-0_16], based on PURE-EV (HansaBioMed Life Sciences) size exclusion chromatography (SEC), followed by centrifugation was used. Five replicates of OF-EVs (n=3 animals/per replicate) and three replicates of UF-EVs (n=5 animals/per replicate) of each experimental group (OF and UF, respectively) were used and the miRNeasy Micro Kit (Qiagen, Hilden, Germany) was used to isolate total RNA, following the manufacturers instructions. The RNA concentration and size distribution were evaluated using an Agilent RNA 6000 Pico kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA was used as input material for the RNA sample preparations. Briefly, 3' and 5' adaptors were ligated to 3' and 5' ends of small RNA, respectively. Then, the first strand cDNA was synthesized after hybridization with reverse transcription primer. The double-stranded cDNA library was generated through PCR enrichment. After purification and size selection, libraries with insertions between 18~40 bp were ready for sequencing on the Illumina sequencing platform Novaseq6000 with SE50. The library was checked with Qubit 2.0 and real-time PCR for quantification and a bioanalyzer 2100 for size distribution detection. Quantified libraries were pooled and sequenced on the Illumina platform Novaseq6000, according to effective library concentration and data amount (20M reads per sample). The sequencing strategy was Single-end 50 bp (SE50). The index-coded samples were clustered according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on the Illumina platform Novaseq6000, and single-end reads were generated.