Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species

Deep sequencing of RNAs has greatly aided the study of the transcriptome, enabling comprehensive gene expression profiling and the identification of novel transcripts. While mRNAs are of the greatest interest in gene expression studies as they encode for proteins, mRNAs make up only 3-5% of total RNAs, with the majority comprising ribosomal RNAs (rRNAs). Therefore, applications of deep sequencing to RNA face the challenge of how to efficiently enrich mRNA species prior to library construction. Traditional methods extract mRNAs using oligo-dT primers targeting the poly-A tail on mRNAs; however, this approach is not comprehensive as it does not account for mRNAs lacking the poly-A tail or other lncRNAs that we may be interested in. Alternative methods deplete rRNAs, but such approaches require species-specific probes and the commercially available kits are costly and have only been developed for a limited number of model organisms. Here we describe a quick, cost-effective method for depleting rRNAs using custom-designed oligos. We use chickens as an example species for probe design, and the same approach also apply to mice. Using this protocol, we have not only removed the rRNAs from total RNAs for RNA-seq library construction but also depleted rRNA fragments from ribosome-protected fragments for ribosome profiling. Currently, this is the only rRNA depletion-based method for avian species; this method thus provides a valuable resource for both the scientific community and the poultry industry.