First insights on miRNAs expression of seminal plasma extracellular vesicles and sperms in ducks of contrasted sperm motility
duck miRNAs from sperm with high (DHS) or low (DLS) sperm motility and seminal plasma extracellular vesicles with high (DHSE) or low (DLSE) sperm motility
Steps to reproduce
Semen collection and evaluation Semen samples from ducks were collected by trained professionals.Sperm viability (%) was assessed using Trypan Blue to identify the proportion of live and dead sperms. seminal plasma extracellular vesicles(SPEVs) and sperms sample preparation and isolation Samples of sperms and SPEVs with high and low sperm motility (named DHS/DLS and DHSE/DLSE, respectively) were separated by the ultrahigh-speed differential centrifugation method. Centrifuged at 1000×g for 10 min to collect sperm cells, SPEVs was collected after ultracentrifuged twice at 100,000×g. RNA Isolation, small RNA library construction, and sequencing SPEVs RNA was extracted using the Total Exosome RNA and Protein Isolation Kit (Thermo Fisher, CA, USA) according to the manufacturer’s instructions. Trizol (Invitrogen, CA, USA) was used to isolate total RNA from sperms.Libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina Inc., CA, USA), and RNA sequencing was carried out on an Illumina HiSeq 4000 platform. Data analysis To obtain the clean reads, FastQC tools (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) were used to remove the low-quality reads and trimming adapter sequences. The miRNAs were identified based on the annotation information of the Anas platyrhynchos genome (https://www.ncbi.nlm.nih.gov/assembly/GCF_015476345). employed the DESeq R package (1.24.0) to identify significant differential expressed (DE) miRNAs between the libraries.