Mastering Human and Bacterial rRNA Depletion
In high-sensitivity RNA analyses, such as RNA-Seq or metatranscriptomics, reducing the proportion of ribosomal RNA (rRNA) in your sample is absolutely critical. In particular, it is common to need to remove both human and bacterial rRNA at the same time. In this post, I’ll walk you through exactly how to efficiently deplete human + bacterial rRNA in a single reaction using the NEBNext v2 rRNA Depletion Kit, and how you can design species-specific custom probes to target any organism of interest.
1. Why rRNA Depletion Matters
Most biological samples contain 80–90% rRNA as part of their total RNA. If you sequence without removing that rRNA, the reads will be dominated by ribosomal transcripts, and your mRNA, noncoding RNAs, or microbial RNAs of interest will be buried in the noise.
- Without rRNA depletion: low usable (informative) read count, high background
- With effective rRNA depletion: deeper transcriptome coverage, cost savings, better accuracy
Especially when you have a human tissue sample that also contains a microbiome or potential pathogens, you need to remove both human and bacterial rRNA to accurately capture gene expression profiles or microbial community composition.
2. NEBNext v2 rRNA Depletion Kit: Simultaneous Human + Bacterial Removal
NEB’s NEBNext® v2 rRNA Depletion Kit is designed to let you remove both human and bacterial rRNA in a single reaction, saving time and sample handling.
1) Kit Components
- NEBNext v2 rRNA Depletion Solution (contains probes targeting human rRNA): 2 μL
- NEBNext Bacterial rRNA Depletion Solution (contains probes targeting bacterial rRNA): 2 μL
- Simply mix these two solutions (total 4 μL of depletion probes) in one tube to deplete human + bacterial rRNA in one step.
2) Workflow Overview
- Total RNA Extraction (e.g., using Qiagen RNeasy Kit, TRIzol, etc.)
- Combine 2 μL of Human rRNA Depletion Solution + 2 μL of Bacterial rRNA Depletion Solution in your reaction tube.
- Perform the rRNA depletion reaction according to the NEB protocol.
- Purify the rRNA-depleted RNA and proceed to RNA-Seq library prep.
3) Key Advantages
- Single-tube reaction streamlines the process and reduces pipetting steps.
- Saves time and cost (no need to run two separate depletion reactions).
- High depletion efficiency thanks to NEB’s optimized probe design.
3. When You Need Species-Specific rRNA Depletion
If you want to remove rRNA from a specific bacterial species that is not covered by the standard bacterial depletion panel, you can use:
NEBNext® RNA Depletion Core Reagent Set
- This product comes without any probes. Instead, you design and supply your own custom depletion probes.
- Use NEB’s Custom RNA Depletion Design Tool to create probes specific to your organism’s rRNA sequences.
- You can then combine your custom probe pool with the Core Reagent Set’s depletion reaction. You can also mix those custom probes with the Human rRNA Depletion Kit if you need both.
1) Custom Probe Pool Design Steps
- Gather your target species’ rRNA sequences (e.g., from NCBI’s rRNA database).
- Go to NEB’s Custom RNA Depletion Design Tool and input those rRNA sequences to design a set of depletion probes.
- Order the probes (e.g., FITC-labeled or 5′-biotinylated, according to NEB specifications).
- Mix your custom probe pool into the Core Reagent Set’s depletion reaction according to NEB’s recommendations.
2) Custom Probe Usage Guidelines
Refer to NEB’s Guidelines for use of Custom RNA Depletion Probe Pools with the NEBNext RNA Depletion Core Reagent Set for detailed instructions on optimal probe concentration, reaction temperature/time, and purification steps.
4. Checking Bacterial Species Compatibility
Before you buy the standard bacterial depletion kit, you should confirm that your bacterial species of interest is in NEB’s compatibility list.
Visit NEB’s Bacterial rRNA Depletion Kit (Bacteria) Species Compatibility page to see which species are covered.
If your organism isn’t listed, you’ll need to move to the NEBNext RNA Depletion Core Reagent Set approach and design custom probes yourself.
5. Post-Depletion Workflow
1) Verify rRNA Removal
- Run a Bioanalyzer or TapeStation check: you should see 28S/18S rRNA peaks dramatically reduced or gone.
- Quantify total RNA using Qubit or Nanodrop to check how much RNA was lost during depletion.
2) RNA-Seq Library Preparation
- Take the rRNA-depleted RNA into an RNA-Seq library kit (for example, NEBNext Ultra II RNA Library Prep Kit). Perform cDNA synthesis, adapter ligation, and PCR amplification.
- Because most rRNA is gone, your library complexity and quality will improve significantly.
3) Sequencing and Data Analysis
- Sequence on an Illumina or Oxford Nanopore platform.
- Filter out any remaining rRNA reads and focus on non-rRNA reads for gene expression quantification or microbial community profiling.
- Lower rRNA content means more usable reads per sample, reducing overall sequencing costs.
6. FAQ: Frequently Asked Questions
2) GC content and melting temperature (Tm): Keep individual probe Tms similar by adjusting probe length and GC content, so they all hybridize under the same conditions.
3) Probe length: Typically design probes between 50–120 nt. Too short reduces specificity; too long makes temperature optimization tricky.
4) Cross-reactivity: Use BLAST to check that your probes won’t bind off-target to other RNAs in your sample.
7. Final Tips and Recommendations
- During sample preparation, aim for a high RNA Integrity Number (RIN) so that after rRNA depletion, you still have enough high-quality RNA for library prep.
- Follow NEB’s recommended reaction temperature, mixing time, and probe concentration, but be prepared to optimize slightly depending on your sample type (e.g., complex microbial communities).
- When you use custom probes for the first time, run a pilot reaction (just one or two tubes) to confirm depletion efficiency before scaling up.
Further Reading
