Product Class: Kit

NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat)

This product provides superior performance compared to the original E6310 kit. Note that RNA Sample Purification beads are not included in this kit. If beads are required, we recommend ordering the NEBNext rRNA Depletion Kit v2 with Sample Purification Beads (NEB #E7405). 


Catalog #E7400

Product Introduction

The abundance of ribosomal RNAs (rRNAs), constituting 80–90% of total RNA, necessitates removal upstream of next generation sequencing and other applications. This second generation rRNA depletion kit incorporates reagent, probe and protocol improvements, resulting in superior depletion performance. The efficient RNAse-H-based workflow, and close spacing of probes, enables effective depletion from both low- and high-quality RNA, with a broad range of input amounts. 

  • Superior depletion of rRNA from human, mouse and rat RNA
  • Depletion of cytoplasmic (5S, 5.8S, 18S, 28S, human ITS, ETS) and mitochondrial (12S and 16S) rRNA
  • 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.

To request a sample of our NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat), please click here. 

Product Information

Description


Highly expressed transcripts with minimal biological interest, such as ribosomal RNA (rRNA) can dominate readouts, masking detection of more informative low-abundance transcripts. This second-generation NEBNext rRNA Depletion Kit is has been further optimized to incorporate reagent, probe and protocol improvements to the RNaseH-based workflow, resulting in superior depletion performance. 

  • The efficiency of this workflow, and close spacing of probes, enables effective depletion from both low- and high-quality RNA, with a broad range of input amounts from human, mouse and rat samples.
  • The rRNA-depleted RNA can be used in RNA-seq, random-primed cDNA synthesis, or other RNA analysis methods. 
  • The NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) depletes cytoplasmic (5S, 5.8S, 18S, 28S, human ITS, ETS) and mitochondrial (12S, 16S) rRNA.
  • The kit is also available with RNAClean® beads.



Figure 1: The NEBNext rRNA Depletion Kit v2 enriches for RNAs of interest across a wide range of total RNA inputs in human, mouse and rat



Universal human, mouse and rat reference total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75 bp). 10 Million reads were sampled (seqtk) from each library and reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for human baits (5S, 5.8S, 12S, 16S, 18S, 28S, ETS/ITS) or mouse/rat baits (5S, 5.8S, 12S, 16S, 18S, 28S). Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates. The NEBNext rRNA Depletion Kit v2 is efficient at depleting rRNA across species and input amounts.




Figure 2: Treatment with the NEBNext rRNA Depletion Kit v2 does not affect the abundances of non-targeted transcripts



Universal human, mouse and rat reference total RNA (1 µg) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). 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 75bp). 10 million reads were sampled (seqtk) for depleted libraries and 100 million reads for undepleted libraries. GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. Transcript expression is consistent after depletion across species.




Figure 3: Transcript expression correlation is preserved across a wide range of inputs



Universal human, mouse and rat reference total RNA (1 µg) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75bp). GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. Transcript expression correlation is consistent regardless of input amount.




Figure 4: The NEBNext rRNA Depletion Kit v2 efficiently depletes rRNA from degraded FFPE total RNA while preserving transcript abundances



Human adult normal liver tissue FFPE Total RNA, RIN 2.3 (100 ng and 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (A, B, C) or the TruSeq® Stranded Total RNA Gold kit (A). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). 20 Million reads were sampled (seqtk) from depleted libraries and 200 million reads from undepleted libraries. (A) Reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for 5S, 5.8S, 12S, 16S, 18S, 28S and ETS/ITS as baits. Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates and error bars indicate standard error. (B) and (C) GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. The NEBNext kit provides superior depletion of rRNA from FFPE (degraded) samples and offers the flexibility of lower total RNA input amounts. Transcript expression correlation is consistent after treatment and regardless of input amount.




Figure 5: The NEBNext rRNA Depletion Kit v2 depletes both mature and pre-rRNA from human total RNA



Human universal reference total RNA (1 µg) was depleted of rRNA using the original NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310) and the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400). RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75 bp). 10 Million reads were sampled (seqtk) from each library. Reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for 5S, 5.8S 12S, 16S, 18S, 28S and ETS/ITS as baits. Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates. NEBNext rRNA Depletion Kit v2 provide superior depletion of rRNA.




Figure 6: Workflow



This product is related to the following categories:
RNA Depletion & mRNA Enrichment,
Epitranscriptome Analysis,
RNA Library Prep for Illumina,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7400S     -20    
      NEBNext® DNase I E7753-2VIAL -20 1 x 0.015 ml 20,000 units/ml
      NEBNext® Thermostable RNase H E7752-2VIAL -20 1 x 0.012 ml 5,000 units/ml
      NEBNext v2 rRNA Depletion Solution E7401-2VIAL -20 1 x 0.012 ml Not Applicable
      RNase H Reaction Buffer E6312-2VIAL -20 1 x 0.012 ml Not Applicable
      DNase I Reaction Buffer E6315-2VIAL -20 1 x 0.03 ml Not Applicable
      Nuclease-free Water E6317-2VIAL -20 1 x 0.4 ml Not Applicable
      NEBNext Probe Hybridization Buffer E6314-2VIAL -20 1 x 0.012 ml Not Applicable
  • E7400L     -20    
      NEBNext® DNase I E7753-3VIAL -20 1 x 0.06 ml 20,000 units/ml
      NEBNext® Thermostable RNase H E7752-3VIAL -20 1 x 0.048 ml 5,000 units/ml
      NEBNext v2 rRNA Depletion Solution E7401-3VIAL -20 1 x 0.048 ml Not Applicable
      RNase H Reaction Buffer E6312-3VIAL -20 1 x 0.048 ml Not Applicable
      DNase I Reaction Buffer E6315-3VIAL -20 1 x 0.12 ml Not Applicable
      Nuclease-free Water E6317-3VIAL -20 1 x 1.5 ml Not Applicable
      NEBNext Probe Hybridization Buffer E6314-3VIAL -20 1 x 0.048 ml Not Applicable
  • E7400X     -20    
      NEBNext® DNase I E7753-4VIAL -20 1 x 0.24 ml 20,000 units/ml
      NEBNext® Thermostable RNase H E7752-4VIAL -20 1 x 0.192 ml 5,000 units/ml
      NEBNext v2 rRNA Depletion Solution E7401-4VIAL -20 1 x 0.192 ml Not Applicable
      RNase H Reaction Buffer E6312-4VIAL -20 1 x 0.192 ml Not Applicable
      DNase I Reaction Buffer E6315-4VIAL -20 1 x 0.48 ml Not Applicable
      Nuclease-free Water E6317-4VIAL -20 1 x 6 ml Not Applicable
      NEBNext Probe Hybridization Buffer E6314-4VIAL -20 1 x 0.192 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

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

Features

  • Superior depletion of rRNA from human, mouse and rat RNA
  • Depletion of cytoplasmic (5S, 5.8S, 18S, 28S, human ITS, ETS) and mitochondrial (12S and 16S) rRNA
  • 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

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.

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the difference between the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) and the original NEBNext® rRNA Depletion Kit (Human/Mouse/Rat)?
  2. Which species are compatible with the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) for? Will it work for other species?
  3. Which rRNA subunits are depleted with the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat)?
  4. Can I use this product with degraded RNA or fragmented RNA?
  5. What total RNA input should I use?
  6. Why must the RNA be free of DNA?
  7. What is the expected RNA yield recovered after rRNA depletion?
  8. What is the percentage of rRNA remaining after depletion?
  9. How can I determine if the rRNA depletion was efficient?
  10. Does the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) work for ribosomal footprinting or profiling applications?
  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. To remove ribosomal RNA from total RNA, should I use the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or the NEBNext rRNA Depletion Kit v2 (Human, Mouse, Rat)?
  14. Can I use more than one type of NEBNext Depletion Solution in the NEBNext RNA depletion workflow?
  15. 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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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.

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