Comparison of Poly-A⁺ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A⁺ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A⁺ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A⁺ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A⁺ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A⁺ selected libraries. Overall, poly-A⁺ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.