Abstract
RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues.
Highlights
RNA interference (RNAi), is a complex and highly conserved gene regulatory mechanism [1,2] mediated via small RNAs
To investigate the identity of the other abundant ribosomal RNAs (rRNAs) fragments in the accessory gland + testes (AGT) sequencing libraries subjected to the single oligo blocking, we examined the identity of the reads aligning to D. melanogaster rRNA sequences (Fig 3A and 3B)
The results showed that the use of blocking oligos was a highly effective and specific method of eliminating problematic rRNA sequences from library construction
Summary
RNA interference (RNAi), is a complex and highly conserved gene regulatory mechanism [1,2] mediated via small RNAs (sRNAs). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have