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

Catalog # Concentration Size List Price Quantity Your Price
E7400L 24 reactions $1,614.00
$1,452.60
E7400S 6 reactions $444.00
$399.60
E7400X 96 reactions $5,804.00
$5,223.60
Catalog # Size List Price Your Price
E7400L 24 reactions $1,614.00
$1,452.60
E7400S 6 reactions $444.00
$399.60
E7400X 96 reactions $5,804.00
$5,223.60
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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. 


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



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
  NEBNext v2 rRNA Depletion Solution E7401-2VIAL -20 1 x 0.012 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
  NEBNext v2 rRNA Depletion Solution E7401-3VIAL -20 1 x 0.048 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
  NEBNext v2 rRNA Depletion Solution E7401-4VIAL -20 1 x 0.192 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
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
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

Companion Products
Citations
Additional Citations
  • Butler, D.J. et al. (2020) Shotgun transcriptome and isothermal profiling of SARS-CoV-2 infection reveals unique host responses, viral diversification, and drug interactions bioRxiv DOI: https://doi.org/10.1101/2020.04.20.048066
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Specifications
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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]
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