Abstract
Global transcriptomic analysis is often hindered by the high abundance of ribosomal RNA (rRNA) content in bacterial cells, comprising up to 98% of RNA in cells. To remove unwanted rRNA and enrich for valuable protein coding transcripts of interest, rRNA depletion by capture probe hybridization has been the method of choice for RNA-seq, because it maintains relative transcriptome levels. The existing RiboMinus™ Bacteria design has had limited success with partially degraded total RNA, due to an abundance of fragmented rRNA contamination in compromised RNA that the current design of eight locked nucleic acid (LNA) probes does not address. Presently, we evaluate the efficiency of a new RiboMinus™ Bacteria 2.0 Kit in depleting rRNA from various inputs (100ng-5ug) of E. coli Total RNA, which utilizes a new probe mix design. The RiboMinus™ procedure entails hybridization of a new set of 3â€2-biotin labeled ssDNA oligonucleotide probes (called RiboMinus™ Pan-Prokaryote Probe Mix) targeting sequence-specific regions of 16S, 23S rRNA and 5S rRNA. This RNA/probe hybrid is then removed from the sample using streptavidin-coated magnetic beads, leaving behind rRNA-depleted RNA. Sequencing results indicate that the newly developed RiboMinus™ Pan-Prokaryote Probe Mix in the RiboMinus™ Bacteria 2.0 workflow exhibits superior rRNA depletion efficiency compared to the existing kit, with less than 10% of total reads mapping to 16S and 23S rRNA vs. 90-95% of total reads mapping to rRNA using the existing kit. Removal of rRNA also results in a 10-fold increase of protein coding transcripts, from 5% in the existing kit to 55% in RiboMinus™ Bacteria 2.0. In addition, rRNA removal increases sensitivity for detection of noncoding transcripts. Finally, we also demonstrate that the newly developed RiboMinus™ Bacteria 2.0 Kit utilizing the new RiboMinus™ Pan-Prokaryote Probe Mix exhibits high ribosomal removal efficiency when tested with Salmonella enterica total RNA.