Product Class: Kit

NEBNext® RNA Depletion Core Reagent Set


Catalog #E7865

Product Introduction

Introducing a new solution for customized depletion of abundant RNAs.

In RNA-seq, highly expressed transcripts with minimal biological interest, such as ribosomal RNA (rRNA) can dominate readouts and mask detection of more informative low-abundance transcripts. This challenge is amplified when working with sample types for which pre-designed RNA depletion kits are not available. The NEBNext RNA Depletion Core Reagent set provides a customized approach to deplete unwanted RNA from any organism, using probe sequences designed with the user-friendly NEBNext Custom RNA Depletion Design Tool.

The efficient RNase-H-based workflow, and close tiling of probes designed using the online tool, enables effective depletion from both low- and high-quality RNA, with a broad range of input amounts. 

  • Compatible with a broad range of input amounts: 10 ng - 1µg
  • 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

RNA-seq samples can include a large dynamic range of transcript expression, and unwanted, abundant RNAs must be removed in order to achieve efficient sequencing of the RNAs of interest. While pre-designed kits are available for some sample types (e.g. human, mouse, rat, some bacteria) and abundant RNAs (e.g. rRNA, globin mRNA), a customized approach is required for rRNA removal from other sample types as well as for removal of specific non-rRNA RNAs. 

Customized depletion of unwanted RNAs is enabled by use of the NEBNext RNA Depletion Core Reagent together with custom probes, following this workflow:

STEP 1: Use the online NEBNext Custom RNA Depletion Design Tool to obtain custom probe sequences, by entering the sequence of your target RNA. 

STEP 2: Order ssDNA probe oligonucleotides from your trusted oligo provider. 

STEP 3: Use the probes with the NEBNext Custom RNA Depletion Core Reagent Set or in combination with other NEBNext RNA Depletion Kits.

The NEBNext RNaseH-based RNA depletion workflow together with the closely-tiled custom probes designed using the online tool, allow effective depletion from both low- and high-quality RNA, with a broad range of input amounts.

The kit is also available with RNAClean® beads.

Features

  • Customizable option to deplete unwanted RNA from any organism, using probe sequences designed with the NEBNext Custom RNA Depletion Design Tool.
  • 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

 

Figure 1: NEBNext customized RNA depletion enriches for RNAs of interest by efficiently removing targeted RNA from total RNA across species and a wide range of inputs

The NEBNext Custom RNA Depletion Design Tool was used to design probes against rRNA of the mosquito Aedes aegypti (28S, 18S, 5.8S, 5S, mt16S and mt12S), and the archaeal species Thermococcus kodakarensis and Pyrococcus furiosus (23S, 16S, 5SrRNA1, 5SrRNA2). Total RNA (1 µg, 100 ng or 10 ng) was used as input for rRNA depletion using the RNA Depletion Core Reagent Set with the designed probes. 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 each library. Read pairs were identified as ribosomal using mirabait (6 or more shared 25-mers), and 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 method efficiently depletes targeted rRNA across species and a wide range of total RNA input amount (1 µg–10 ng).



Figure 2: Removal of targeted RNA with NEBNext customized RNA Depletion does not affect the levels of non-targeted transcripts 

The NEBNext Custom RNA Depletion Design Tool was used to design probes against Aedes aegypti rRNA. Total RNA was extracted from adult Aedes aegypti mosquitos (Benzon Research) using the Monarch® Total RNA Miniprep Kit (NEB #T2010S). Total RNA (100 ng and 10 ng) was used as input for rRNA depletion using the NEBNext RNA Depletion Core Reagent Set with the designed probes. 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 from undepleted libraries. Transcript abundances were estimated using Salmon and transcripts from Vectorbase (AaegL5.2 assembly). Read counts and R2 values for the linear fit are shown. A) Treatment does not affect abundances of non-targeted transcripts. B) Transcript abundances are maintained between replicates and across input amounts. 



Figure 3: Combined probe pools efficiently deplete human rRNA and mitochondrial mRNA

The NEBNext Custom RNA Depletion Design Tool was used to design probes against human mitochondrial mRNA. The probes were used in combination with the probe pool from the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). 1 μg of total Universal Human Reference RNA (Agilent®) was depleted of mitochondrial RNA and rRNA using the combined probes. 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 each library. A. Read pairs were identified as ribosomal and mitochondrial using mirabait (6 or more, 25-mers), and levels of rRNA and mtRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. Both rRNA and mitochondrial RNA are efficiently depleted. B. Integrative Genome Viewer (IGV) visualization of read coverage across the human mitochondrial genes.


Figure 4: Workflow

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

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7865S     -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
      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
  • E7865L     -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
      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

Properties & Usage

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

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. How can I design DNA probes to use with the NEBNext RNA Depletion Core Reagent Set? 
  2. Are the probes designed by the NEBNext Custom RNA Depletion Design Tool sense or antisense?
  3. Can the input sequence into the NEBNext Custom RNA Depletion Design Tool contain IUPAC Ambiguity Code?
  4. Does the NEBNext Custom RNA Depletion Design Tool check for probes targeting transcripts other than the ones provided?
  5. I have used NEBNext Custom RNA Depletion Design Tool and obtained probe sequences, what’s the next step?
  6. How do I make a working probe pool to be used with the NEBNext RNA Depletion Core Reagent Set?
  7. What’s the maximum number of probes that can be combined in a pool?
  8. Can I use this product with degraded RNA or fragmented RNA?
  9. What total RNA input should I use with the NEBNext RNA Depletion Core Reagent Set? 
  10. Why must the RNA be free of DNA?
  11. How can I determine if the RNA depletion was efficient?
  12. Can I use Agencourt®AMPure XP Magnetic Beads instead of RNAClean® XP Magnetic Beads/NEBNext® RNA Sample Purification Beads?
  13. Can I use purification methods other than NEBNext® RNA Sample Purification Beads/Agencourt® RNAClean® XP Magnetic Beads?
  14. Does the NEBNext Custom RNA Depletion Tool check for probe redundancy or overlap between target RNA sequences entered?
  15. Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications?

Troubleshooting

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.

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.

Licenses

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.

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information about commercial rights, please email us at gbd@neb.com While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
 

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