Description

RiboCop rRNA Depletion Kits

RiboCop rRNA Depletion Kits enable removal of ribosomal RNA from total RNA and are suited for Next Generation Sequencing as well as other demanding RNA analysis applications.

NEW! Now also available for bacteria.

Broad Species Compatibility

RiboCop rRNA Depletion Kits are available for Human/Mouse/Rat (Cat. No. 037) and for Bacteria (Cat. No. 125 – 127).

Efficient Elimination of rRNA

RiboCop allows efficient removal of rRNAs. RiboCop for Human/Mouse/Rat (HMR, Cat. No. 037) removes cytoplasmic 28S, 18S, 5.8S, 45S, 5S, as well as mitochondrial mt16S, mt12S ribosomal RNA sequences in human, mouse, and rat samples. RiboCop for Bacteria removes 23S, 16S, and 5S rRNA from Gram positive and Gram negative bacterial species as well as from mixed bacterial samples. Sequencing depth is thus preserved for relevant RNA species.

Simple Workflow

The RiboCop protocol takes 1.5 hours to complete (30 minutes hands-on time). No enzymatic reactions or mechanical shearing steps are involved, leaving full-length transcripts intact for downstream processing.

Flexible Input Requirements

RiboCop is suitable for rRNA depletion of 1 – 1,000 ng total RNA input amounts and is compatible with intact or degraded RNA (including FFPE) samples. Specific input recommendations apply for certain sample types and are described in the respective User Guides.

Automation

Magnetic beads are used for depletion and purification steps rendering the protocol completely automation-friendly.

Various Downstream Applications

RiboCop rRNA Depletion Kits are offered as stand-alone kits in different kit sizes for use with any downstream application. For convenience, RiboCop for Human/Mouse/Rat (HMR) is also available in combination with the CORALL Total RNA-Seq Library Prep Kit (Cat. No. 096).
For questions regarding RiboCop-compatibility with other species, please contact support@lexogen.com.

Workflow

Samples are treated using a set of affinity probes for specific depletion of rRNA sequences. RiboCop probes efficiently remove rRNA and therefore afford a comprehensive view of transcriptome composition.

Denaturation of total RNA
Step 1:
Affinity probes and total RNA are mixed and denatured.
ribocop_workflow_01Timer_RiboCop_V1.3
Hybridization of affinity probes to rRNA
Step 2:
Denatured total RNA and probes are hybridized.
ribocop_workflow_02Timer_RiboCop_V1.3
Removal of bound rRNA molecules
Step 3:
Depletion beads are conditioned and used to remove probes
along with hybridized ribosomal RNA from solution.
Timer_RiboCop_V1.3
Purification of depleted RNA
ribocop_workflow_04Timer_RiboCop_V1.3
Step 4:
The depleted RNA is purified for downstream processing.

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_01_RiboCop-Bacteria-Barplot-depletion-over-input-range-1.jpg

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-02_Unbiased expression profile maintenance

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).

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.

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)

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.

Excellent Reproducibility and Depletion without of 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.

FAQ

Frequently Asked Questions

Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact support@lexogen.com.

If you don´t have a thermomixer available you can just run it on a thermocycler without agitation.
RiboCop rRNA depleted total RNA is suitable to be used with the CORALL Total RNA-Seq Library Prep Kit. Please find more information about this bundle in the CORALL Total RNA-Seq Library Prep Kit User Guide.
RiboCop HMR contains probes for the rRNA depletion of human, mouse and rat RNA samples and was also successfully tested on hamster, mini pig, and zebrafish samples.
Depending on input sample, standard recovery rate yield varies from 1 – 3%.
Depending on input sample, depletion down to 1 – 2% is standard.
RiboCop HMR is suitable for input amounts from 1 ng – 1 µg of total RNA input.
RiboCop HMR V1.3 contains the new, faster protocol allowing for rRNA depletion in 1.5 hours (30 minutes hands-on time). The kit components and performance have not changed. If you wish to perform the V1.2 protocol, you can do so. V1.3 and V1.2 are fully compatible.

The complete list of changes can be found in the Revision History of the RiboCop V1.3 User Guide.

For rRNA depletion from Gram negative (e.g., E. Coli) or Gram positive (e.g., B. Subtilis) bacterial monocultures we recommend the RiboCop for Gram Negative or Gram Positive Bacteria kits, respectively (Cat. No. 126 or 127).

The META Probe Mix (Cat. No. 125) is primarily suited for depletion of total RNA from mixed bacterial samples (e.g., for meta-transcriptome analysis of complex communities or microbiome samples).

The recommended input range for efficient depletion with RiboCop rRNA Depletion Kits for Bacteria is 1 – 1,000 ng of total RNA for monocultures of Gram negative or Gram positive bacteria using the respective G- and G+ Probe Mixes (Cat. No. 126 and 127, respectively). We suggest initial experiments to be performed using 100 ng of input material.

The META Probe Mix (Cat. No. 125) is primarily suited for depletion of 1 – 1,000 ng of total RNA from mixed bacterial samples. The META Probe Mix may also be used at 1 – 100 ng when working with monocultures.

Processing high quality RNA from bacterial monocultures with the Gram Positive or Gram Negative Probe Mixes usually results in less than 1 % remaining rRNA reads.

Depletion rates for the META Probe Mix using RNA from mixed bacterial samples can vary depending on the sample. For specific questions please contact support@lexogen.com.

Depletion rates may vary depending on the quality of the RNA and purity of the sample. Contaminants, such as salts, metal ions and organic solvents, may have a negative impact on the efficiency of the protocol.

Yes, however the recommended input range in this case is 1 – 100 ng of total RNA.

Downloads

RiboCop Free Trial Kit Form

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If you would like to receive a RiboCop Free Trial Kit, please fill in the form.

If you have any questions, please do not hesitate to contact us at info@lexogen.com or +43 (0) 1 345 1212 – 41.

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