The astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed.
Researchers from the University of Bayreuth describe a workflow for the efficient depletion of rRNA in the planarian model species S. mediterranea. Their protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. The researchers tested their approach on total RNA isolated from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium.
Efficiency of rRNA removal from total planarian RNA
a Schematic representation of rRNA depletion workflow. Biotinylated DNA probes are hybridized to rRNA, followed by subtraction of DNA-rRNA hybrids using streptavidin-coated magnetic beads. b Separation profile of planarian total RNA. The large peak at 1527 nts corresponds to the co-migrating 18S rRNAs and the two fragments of processed 28S rRNA. LM denotes the lower size marker with a length of 15 nts. c Increasing concentration of NaCl improves the efficiency of rRNA removal. d Total planarian RNA after rRNA depletion. e Removal of DNA-rRNA hybrids was performed in two consecutive steps using streptavidin-coated magnetic beads resuspended in 2x of 1x B&W buffer
The ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications this procedure might be adapted to any prokaryotic or eukaryotic species of choice.