A large number of methods are available to deplete ribosomal RNA reads from high-throughput RNA sequencing experiments. Such methods are critical for sequencing Drosophila small RNAs between 20 and 30 nucleotides because size selection is not typically sufficient to exclude the highly abundant class of 30 nucleotide 2S rRNA. Here we demonstrate that pre-annealing terminator oligos complimentary to Drosophila 2S rRNA prior to 5′ adapter ligation and reverse transcription efficiently depletes 2S rRNA sequences from the sequencing reaction in a simple and inexpensive way. This depletion is highly specific and is achieved with minimal perturbation of miRNA and piRNA profiles.
High-throughput RNA sequencing (RNA-seq) has revolutionized our understanding of RNA biology, but the presence of high abundance RNA species that are not of direct interest can be problematic. For this reason, there is great interest in developing methods that enrich specific classes of RNA for RNA-seq applications. Historically, the most commonly used method for enriching mRNA depends on the presence of poly(A) tails. However, this method is not applicable in non-eukaryotes lacking polyadenylation; it is also incapable of enriching for many non-coding RNAs and degraded RNAs that lack poly(A) tails. In contrast to enriching for poly(A), an alternative approach is to directly deplete nuisance classes of RNA. A recent survey compared different methods for depleting nuisance species of rRNA, favoring rRNA hybridization to DNA oligos and removal of RNA-DNA hybrids by RNase H treatment.
Here researchers from the University of Kansas present a simple method for eliminating 2S RNA from small RNA sequencing reactions in Drosophila with significant time and cost saving. This method may also have broader applications for other forms of RNA-seq.
For sequencing small RNAs (miRNAs, siRNAs and piRNAs), enrichment is usually achieved by size selection. Since these classes of RNA are typically between 20 and 30 nt, size selection from total RNA, followed by ligation, PCR, and further size selection, generally achieves good results In Drosophila, however, size selection is not typically sufficient because the highly abundant class of 2S rRNA is 30 nt long. To deal with this problem, two approaches have been developed. One method, using RNase H, employs hybridization with DNA oligos complementary to 2S rRNA and directed degradation. A second method depletes 2S rRNA by hybridization using oligos bound to magnetic beads, from which non-2S rRNA is removed off as supernatant. Both methods have proven effective in eliminating 2S rRNA reads, but each requires additional steps and reagents which, in the case of magnetic beads, can be costly. We therefore wanted to determine if depletion could be achieved during 5′ ligation and cDNA synthesis by specifically blocking 2S RNA products from jointly participating in these two reactions. For 2S RNA, this is expected to yield truncated cDNA products lacking sequences complementary to the 5′ adapter. Without these sequences, library PCR amplification is impossible. Blocking with terminator oligos may thus serve as a simple means to eliminate 2S rRNA from the sequencing reaction without additional enzymatic steps or use of magnetic beads.
Supplementary Protocol (.pdf)