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NEBNext® RNA Depletion Core Reagent Set

Catalog # Concentration Size List Price Quantity Your Price
E7865L 24 reactions $1,393.00
$1,253.70
E7865S 6 reactions $383.00
$344.70
E7865X 96 reactions $5,018.00
$4,516.20
Catalog # Size List Price Your Price
E7865L 24 reactions $1,393.00
$1,253.70
E7865S 6 reactions $383.00
$344.70
E7865X 96 reactions $5,018.00
$4,516.20
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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.

 

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

 
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  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
  DNase I Reaction Buffer E6315-2VIAL -20 1 x 0.03 ml
  Nuclease-free Water E6317-2VIAL -20 1 x 0.4 ml
  NEBNext Probe Hybridization Buffer E6314-2VIAL -20 1 x 0.012 ml
  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
  DNase I Reaction Buffer E6315-3VIAL -20 1 x 0.12 ml
  Nuclease-free Water E6317-3VIAL -20 1 x 1.5 ml
  NEBNext Probe Hybridization Buffer E6314-3VIAL -20 1 x 0.048 ml
  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
  RNase H Reaction Buffer E6312-4VIAL -20 1 x 0.192 ml
  DNase I Reaction Buffer E6315-4VIAL -20 1 x 0.48 ml
  Nuclease-free Water E6317-4VIAL -20 1 x 6 ml
  NEBNext Probe Hybridization Buffer E6314-4VIAL -20 1 x 0.192 ml
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

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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.

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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