Product Class: Kit

NEBNext® rRNA Depletion Kit (Bacteria)


Catalog #E7850

Product Introduction

Ribosomal RNA (rRNA) is highly abundant in bacterial samples, and its removal is desirable in order to reveal the biological significance of less abundant transcripts. Specific enrichment of bacterial mRNAs is challenging due to their lack of poly(A) tails, and so the converse - efficient and specific removal of bacterial rRNA – is necessary.

The NEBNext® rRNA Depletion Kit (Bacteria) employs the NEBNext RNase H-based RNA depletion workflow to target removal of rRNA from gram-positive and gram-negative organisms. The method is effective with intact and degraded RNA, from monocultures or samples with mixed bacterial species (e.g., metatranscriptomic).

  • Efficient, specific depletion of bacterial rRNA (5S, 16S, 23S)
  • Compatible with both gram-positive and gram-negative organisms
  • Effective with monocultures and mix of bacterial species (e.g., metatranscriptome)
  • Compatible with a broad range of input amounts: 10 ng - 1 µg
  • Suitable for low-quality or high-quality RNA
  • Fast workflow: 2 hours, with less than 10 minutes hands-on time

The kit is also available with RNAClean® beads.

Product Information

Description

The great majority of RNA in bacteria is ribosomal RNA (rRNA). This highly abundant RNA can conceal the biological significance of less abundant transcripts, and so its efficient and specific removal is desirable. 

The NEBNext® rRNA Depletion Kit (Bacteria) employs the NEBNext RNase H-based RNA depletion workflow to deplete rRNA (5S, 16S, and 23S) from gram-positive and gram-negative organisms. View species tested to date.

The kit is effective with both intact and degraded RNA preparations, from monocultures or samples with mixed bacterial species (e.g., metatranscriptomic).

The kit is also available with RNAClean® beads.


Features

  • Efficient, specific depletion of bacterial rRNA (5S, 16S, 23S)
  • Compatible with both gram-positive and gram-negative organisms
  • Effective with monocultures and mix of bacterial species (e.g., metatranscriptome)
  • Compatible with a broad range of input amounts: 10 ng - 1 µg
  • Suitable for low-quality or high-quality RNA
  • Fast workflow: 2 hours, with less than 10 minutes hands-on time
  • Includes NEBNext RNA Sample Purification Beads (Agencourt® RNAClean® XP)

For use with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) (NEB #E7416), refer to the Protocols tab for UMI Adaptors-specific guidance.
Figure 1: Depletion of ribosomal RNA using the NEBNext rRNA Depletion Kit (Bacteria) enriches for RNAs of interest across a mock community of bacterial species and a range of input amounts

Total RNA was extracted from a lyophilized pool of 20 different bacterial organisms (ATCC® #MSA-2002). Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (Bacteria). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina®, followed by paired-end sequencing (2 x 75 bp). Reads were aligned (Hisat2) to a composite reference genome containing the best matching strains in the NCBI genome database.  Alignments were duplicate marked (Picard) and assessed for transcript levels (ht-seq count). Effective depletion of sequences overlapping with annotated rRNA regions was observed at 100 ng and 10 ng of input RNA for most of the organisms.



Figure 2: Depletion of ribosomal RNA with NEBNext enriches for RNAs of interest across monoculture species

Total RNA (100 ng) from Escherichia coli and Clostridum phytofermentans was depleted of rRNA using the NEBNext rRNA Depletion Kit (Bacteria). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina®, followed by paired-end sequencing (2 x 75 bp). Reads were aligned to each reference genome (Hisat2), duplicate marked (Picard) and assessed for transcript levels (ht-seq count). Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. Effective depletion of sequences overlapping with annotated rRNA regions was observed for all species. 



Figure 3: NEBNext demonstrates consistent depletion of ribosomal RNA across a range of input amounts

E.coli total RNA (1 µg, 100 ng, 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit (Bacteria).  RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina®, followed by paired-end sequencing (2 x 75 bp). Reads were aligned to the E.coli MG1655 reference genome (Hisat2), duplicate marked (Picard) and assessed for transcript levels (ht-seq count). Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. Effective depletion of sequences overlapping with annotated rRNA regions was observed at 1 µg, 100 ng and 10 ng of input RNA. 



Figure 4. NEBNext maintains consistent transcript expression correlation after depletion and across a range of inputs: E. coli



E.coli total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit (Bacteria).  RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina®, followed by paired-end sequencing (2 x 75 bp). 4 Million read pairs were sampled (seqtk) from each library, mapped to the E. coli MG1655 reference genome (Bowtie 2.3.2) before counting reads on genes (htseq-count) and correlating their levels. Correlation analysis of the transcripts indicates consistent transcript expression regardless of treatment or input amount.



Figure 5. NEBNext maintains consistent transcript expression correlation after depletion and across a range of inputs: Mock bacterial community 

Total RNA was extracted from a lyophilized pool of 20 different bacterial organisms (ATCC® #MSA-2002). Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (Bacteria). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina®, followed by paired-end sequencing (2 x 75 bp). 4 Million read pairs were sampled (seqtk) from each library, mapped to a composite genome (Bowtie 2.3.2) before counting reads on genes (htseq-count) and correlating their levels. Correlation analysis of the transcripts indicates consistent transcript expression regardless of treatment or input amount.



Figure 6: NEBNext rRNA Depletion Kit Workflow

This product is related to the following categories:
RNA Depletion & mRNA Enrichment Products,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation Products,

Properties & Usage

Materials Required but not Supplied

  • Magnetic rack (NEB #S1515) or magnetic plate (Alpaqua. cat. #A001322) or equivalent
  • 80% Ethanol (freshly prepared)
  • Thermocycler
  • Any thin wall 200 μl PCR tubes and 1.5 ml tubes
  • Bioanalyzer, Tapestation. (Agilent Technologies, Inc.) or similar instrument and consumables
  • Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987)

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using NEBNext® RNA Library Prep reagents?

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Usage & Guidelines

FAQs & Troubleshooting

FAQs

  1. Which bacterial rRNA subunits are depleted with the NEBNext rRNA Depletion Kit Bacteria
  2. Can the NEBNext® rRNA Depletion Kit (Bacteria) be used on metatranscriptomic samples?
  3. How can I remove rRNA from a mix of mammalian and bacterial samples
  4. What is the percentage of bacterial rRNA remaining after depletion?
  5. How can I determine if the rRNA depletion was efficient?
  6. Can I use this product with degraded RNA or fragmented RNA?
  7. What is the total RNA input I should use?
  8. Why must the RNA be free of DNA?
  9. Which microbiome community was used in the depletion experiments described, and which references were used to assess the performance of the NEBNext® rRNA Depletion Kit (Bacteria) with this sample?
  10. Which species are compatible with the NEBNext® rRNA Depletion Kit (Bacteria)?
  11. Can I use Agencourt® AMPure® XP Magnetic Beads instead of NEBNext® RNA Sample Purification Beads (RNAClean® XP Magnetic Beads)?
  12. Can I use purification methods other than NEBNext® RNA Sample Purification Beads/Agencourt® RNAClean® XP Magnetic Beads?
  13. Which species have been tested with the NEBNext rRNA Depletion Kit (Bacteria)?
  14. Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.