Data Quality Control and Assembly

To ensure the quality of further analytical data, raw reads were filtered using in-house Perl scripts (Annoroad Gene Technology Co., Ltd, Beijing, China) to obtain high-quality reads. The filtering step involved deleting reads with low quality (Phred quality score <5%), adapter contamination, matches to rRNA sequence, or a rate of ambiguous bases higher than 5% (32). The Phred quality score refers to the rate of sequencing errors for a given base; for example, Q30 indicates that the base sequencing error rate was 0.1%. We aligned paired-end clean reads to the oar4.0 sheep reference genome (https://www.ncbi.nlm.nih.gov/assembly/GCF_000298735.2) using HISAT2 (v.2.0.5) (33) with the parameters “–rna-strandness RF” and “–dta -t -p 4”. Only the uniquely mapped reads were assembled, and the expression levels were predicted using String Tie (v.1.3.2d) (33) with the parameter “-G ref.gtf -rf -1”. Thereafter, we calculated the number and ratio of the uniquely mapped reads within the three gene functional elements: exons, introns, and intergenic elements. Homogeneity analysis was subsequently performed to guarantee that the sequencing results did not impact further transcriptome analysis. Thus, mRNA and known lncRNA transcripts were identified.