An efficient one-step rRNA depletion method for RNA sequencing in non-model organisms

RNA sequencing (RNA-seq) has revolutionized global transcriptomic analysis, ribosome footprinting, and polysome profiling, providing a wealth of data. Importantly, many RNA-based omics approaches typically involve either the removal of ribosomal RNA (rRNA) or selection of messenger RNA (mRNA) prior to sequencing, thereby enriching reads that map to the translationally active part of the transcriptome. Prokaryotic mRNA differs from eukaryotic mRNA in that it lacks the 3’ polyadenylated tail, which excludes the use of poly(A)-based selection methods. While commercial rRNA depletion products exist for a growing number of prokaryotes, their proprietary nature and potential inefficiency with non-model organisms are factors that may limit broad-scale application. To mitigate this issue, we designed DepStep, a consolidated workflow for one-step rRNA depletion using species-specific biotinylated antisense probes for selective hybridization and removal of the target rRNA molecules. As a proof-of-concept, RNA-seq libraries of the psychrophilic gram-negative bacterium Shewanella glacialimarina TZS-4 _T were prepared using both DepStep and a commercial rRNA depletion kit for gram-negative bacteria, to which DepStep was benchmarked. DepStep compares favorably to the commercial depletion kit; it efficiently removes >98.6% of the rRNA content, and a slight increase in total read counts aligning to the coding sequences (CDS) was observed. Importantly, DepStep’s cost-per-sample is three times lower than the commercial kit, establishing DepStep as a simple yet cost-effective alternative to commercial solutions.