title: RNA Sequencing of miRNA Profiles in the Ovaries of Small Tail Han Sheep
design: The study followed ethical guidelines approved by the Animal Sciences Institute, CAAS, and complied with ARRIVE guidelines. Ovarian tissues were collected from six healthy, 2-year-old female sheep, with three individuals representing each breedSmall Tail Han (X_LC) and Dolang (D_LC). The animals were housed under standard feeding conditions with unrestricted access to food and water. Human slaughter was conducted by professionals, employing electrical stunning prior to decapitation to minimize suffering. Immediately after excision, ovaries were preserved in liquid nitrogen at -80C. Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) and assessed for quality with a Nanodrop 2000 and an Agilent 2100 Bioanalyzer. Small RNA libraries were prepared using Illumina TruSeq kits, incorporating 3 and 5 adaptor ligation, followed by cDNA synthesis, amplification, and validation. Library integrity was confirmed with the Bioanalyzer 2100 RNA-6000 Nano Kit. Sequencing was performed on an Illumina HiSeq 2500 platform, and raw reads underwent quality control using cutadapt, fastx_toolkit, and NGSQCToolkit. Clean reads were annotated with Rfam v.10.1 and aligned to GenBank and miRBase v.22 to identify known miRNAs. Novel miRNAs were predicted by aligning unmapped reads with cattle and goat miRBase using the BWA algorithm. Expression levels were quantified in transcripts per million (TPM) with upper-quartile normalization. Differential expression analysis was conducted using DESeq2, identifying differentially expressed miRNAs (DEmiRNAs) with a significance threshold of p < 0.05.