RNA extracted from bacterial species comprises up to 98 % of ribosomal RNAs (rRNA), preventing the in-depth analysis of RNAs of interest, particularly in the context of Next Generation Sequencing (RNA-Seq). Moreover, depletion of rRNA derived from multi-species bacterial communities present in soil samples, biofilms, clinical samples (biopsies, stool, urine), surface swaps, etc, poses a unique challenge due to the species-specific rRNA sequence diversity. An efficient rRNA depletion kit can address this variability, and while not all bacterial species will be depleted for their rRNA to the same degree, the sensitivity of the RNA-Seq analysis is greatly enhanced, increasing both, the number of RNAs detected and the confidence in expression values (Westreich et al., 2016).
Lexogen has developed a new RiboCop rRNA Depletion Kits for Bacteria, which efficiently remove 23S, 16S, and 5S rRNA from mixed bacterial samples and monocultures. RiboCop is offered as a stand-alone kit with the option to choose from three optimized Probe Mixes for depletion of rRNA from mixed bacterial samples (META, Cat. No. 125) and from Gram negative or Gram positive bacteria (G- or G+, Cat. No. 126 and 127, respectively) grown in monoculture.
Intact as well as degraded material (including FFPE) may be processed using the time-efficient and simple RiboCop workflow. Resulting rRNA-free samples are suitable for almost any downstream RNA analysis methods including RNA-Seq library preparation approaches focusing on polyadenylated and non-polyadenylated mRNAs and ncRNAs, e.g. Lexogen’s CORALL Total RNA-Seq Library Prep Kit (Cat. No. 095) or shorter RNAs (sRNAs, miRNAs, etc) and adapter ligation, e.g. Lexogen’s Small RNA-Seq Library Prep Kit (Cat. No. 052).
RiboCop uses a set of affinity probes designed for specific depletion of rRNA sequences. Lexogen’s sophisticated probe design allows to minimize off-target effects that can distort NGS data quality. Total RNA input amounts as low as 1 ng and up to 1 μg can be used, depending on sample composition and the chosen depletion Probe Mix. The RiboCop protocol does not include any enzymatic reactions or mechanical shearing steps, leaving full-length transcripts intact for downstream processing. The entire protocol is automation-friendly as magnetic beads are utilized for depletion and purification probes.
Thus, the users of RiboCop for Bacteria will benefit from:
- Efficient depletion of 23S, 16S, and 5S rRNA from monocultures and mixed bacterial samples
- Fast and simple protocol: 1.5 hours with only 30 minutes hands-on time
- Workflow without enzymatic reactions, thus preserved full-length RNA
- Opportunity to use 1 ng – 1 µg total RNA input
- Universal protocol for high- and low-quality RNA
- Sophisticated probe design to eliminate off-target effects
- Excellent protocol reproducibility
Source – Lexogen