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

Abstract

Deep sequencing of RNAs has greatly aided the study of the transcriptome, enabling comprehensive gene expression profiling and the identification of novel transcripts. While messenger RNAs (mRNAs) are of the greatest interest in gene expression studies as they encode for proteins, mRNAs make up only 3 to 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 capture mRNAs lacking the poly-A tail or other long non-coding RNAs that we may be interested in. Alternative mRNA enrichment 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, using chickens as an example species for probe design. With this optimized 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.

Keywords: RNA-seq; Ribo-seq; chickens; mitochondrial rRNA; rRNA.