RiboCop for Bacteria
RNA extracted from bacterial species comprises up to 98 % of the ribosomal RNAs (rRNA), presenting a unique challenge especially when analyzing the transcriptome capacity from complex bacterial communities. Lexogen’s RiboCop rRNA Depletion Kits for Bacteria 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) or from Gram negative or Gram positive bacteria (G- or G+, Cat. No. 126 and 127, respectively) grown in monoculture.
RiboCop is compatible with all random primed library prep kits such as the CORALL Total RNA-Seq Library Prep Kit (Cat. No. 095)or adapter ligation protocols such as the Small RNA-Seq Library Prep Kit (Cat. No. 052).
Robust Performance Over a Wide Range of Input Amounts while Maintaining Unbiased Transcript Abundance
Ribosomal RNA was depleted from E. coli total RNA using RiboCop for Gram Negative Bacteria over a wide range of input amounts (1 ng to 1 µg). RiboCop for Bacteria efficiently reduces rRNA reads from 98 % to 1-3 % over a broad range of input amounts (Fig. 1) while maintaining unbiased transcript abundance (Fig. 2).
Figure 1 | RiboCop rRNA Depletion for Bacteria efficiently removes rRNA across a wide range of input amounts. NGS libraries were prepared using Lexogen’s CORALL Total RNA-Seq Library Prep Kit. Successful depletion was monitored by sequencing and subsequent analysis of remaining rRNA reads from untreated (Total RNA) and depleted E. coli RNA (1, 10, 100 ng and 1 µg). Sequencing on Illumina NextSeq (1×75 bp) and analysis by mapping to the MG1655 reference using BBMap. The percentage of reads mapping to rRNA is plotted in blue.
Figure 2 | RiboCop maintains unbiased expression profiles while efficiently removing undesired ribosomal RNA. Correlation of transcript abundance in untreated samples vs. samples depleted with RiboCop for Gram Negative Bacteria for 1 ng – 1 µg E. coli RNA input. Libraries were prepared using Lexogen’s CORALL Total RNA-Seq Library Prep Kit and sequenced on Illumina NextSeq (1×75 bp). Reads were mapped against the MG1655 reference genome using STAR aligner and counted with FeatureCounts (including multi mappers).
Performance on Meta-transcriptome Samples
The RiboCop rRNA Depletion Kit for Mixed Bacterial Samples (META) is specifically designed for depletion of complex, mixed populations such as environmental communities or microbiome samples. Low quality microbiome samples from stool extractions were used for characterization of the Probe Mix designed for meta-transcriptomics (Tab. 1). The META Probe Mix can also be used for efficient and robust depletion of monocultures up to 100 ng total RNA input (Fig. 3) while maintaining unbiased transcript expression (Fig. 4).
|Sample||% rRNA reads|
|stool microbiome 10 ng||14.75 (± 1.4)|
|stool microbiome 100 ng||15.32 (± 1.4)|
|stool microbiome 1 µg||23.37 (± 1.6)|
Table 1 | Depletion rates for meta-transcriptome analyses. Different amounts of stool microbiome RNA was treated with RiboCop for Mixed Bacterial Samples (META). NGS-Libraries were prepared, sequenced, and analyzed as described in Fig. 1. Reads were mapped to ~60 annotations.
Figure 3 | RiboCop rRNA Depletion for Mixed Bacterial Samples (META) efficiently removes rRNA from various bacterial species. Two RNA amounts from monocultures of the indicated species were subjected to rRNA depletion using the META Probe Mix. Library preparation and sequencing were performed as described in Fig. 1. Reads were mapped against the respective genomes of E. coli MG1655, P. aeruginosa PAO1, and B. subtilis 168. The percentage of reads mapping to rRNA is plotted in blue.
Figure 4 | RiboCop META maintains constant transcript abundance across various species. Correlation of transcript abundance in untreated samples vs. samples depleted with RiboCop for Mixed Bacterial Samples (META) for 100 ng RNA input of three different species. Library preparation, sequencing, and data analysis were performed as described in Fig. 2. Reads were mapped against the E. coli MG1655, P. aeruginosa PA01, or B. subtilis 168 reference genomes, respectively.
We have extensively tested Lexogen’s RiboCop META rRNA depletion kit on bacteria for transcriptomics. Due to a research focus in infection biology at our university, we are working on RNA sequencing projects with various types of bacteria. The RiboCop META is a versatile tool with very good depletion results for all types of bacteria tested so far. Therefore, we would like to continue to use it for future transcriptome profiling projects.
Excellent Reproducibility and Depletion Without Off-target Effects
RiboCop protocols are robust and highly reproducible. Correlation plots show excellent reproducibility between replicates (Fig. 5) independent of the species or the Probe Mix used for depletion.
Figures 2 and 4 additionally show a high correlation between transcript abundance in untreated vs. ribo-depleted samples for a wide input range (1 ng to 1 µg, Fig. 2) as well as across various species (Fig. 4) without outliers. This indicates that depletion with RiboCop for Bacteria occurs without of off-target effects.
Figure 5 | Excellent reproducibility between replicates. Correlation of replicates for two independent depletion reactions using 10 ng total RNA of the indicated species. Total RNA was ribo-depleted with RiboCop for Gram Negative or Gram Positive Bacteria, respectively. Library preparation, sequencing, and data analysis were performed as described in Fig. 4.