Product Class: Kit

NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®

For use in conjunction with this kit, the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) now has an Express protocol option for substantial time savings and a more streamlined workflow.

Note that Sample Purification beads are not included in this kit. If beads are required, we recommend ordering the NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads (NEB #E7765).

Product Introduction

Even more from less, for RNA

Do you need increased sensitivity and specificity from your RNA-seq experiments? Do you have ever-decreasing amounts of input RNA? To address these challenges, our next generation of strand-specific RNA library prep kits have been reformulated at each step, resulting in several fold higher yields of high quality libraries and enabling use of lower input amounts and fewer PCR cycles. The Ultra II Directional RNA kit uses the “dUTP method” (1, 2) for strand-specificity, and has a streamlined, automatable workflow.


Catalog # Size Concentration
E7760S 24 reactions
E7760L 96 reactions

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Product Information

Description


The Ultra II Directional RNA Library Prep Kit for Illumina delivers significantly increased sensitivity and specificity from your RNA-seq experiments, from ever-decreasing amounts of input RNA. In conjunction with ribosomal RNA (rRNA) depletion or poly(A) enrichment, the kit enables the production of high quality libraries from 10 ng of Total RNA, respectively, up to 1 µg.

Strand-specific/directional methods for sequencing RNA provide information on the DNA strand from which the RNA strand was transcribed. This is useful for many reasons including: Identification of antisense transcripts, determination of the transcribed strand of noncoding RNA, and measurement of expression levels of coding or noncoding overlapping transcripts. Overall, the ability to determine the originating strand can substantially enhance the value of a RNA-seq experiment.

The NEBNext Ultra II Directional RNA Library Prep Kit derives its directionality from the “dUTP” method for strand-specificity, with proven superiority for this application.

See what customers are saying about NEBNext Ultra II Directional RNA.

Features

Also available with optional SPRIselect® beads for clean-up and size-selection steps.

Please note that adaptors, primers, rRNA depletion reagents and poly(A) mRNA isolation reagents are not included in the kit and are available separately.

For extensive NEBNext Ultra II performance data, click the links in the Features above and download our technical note for poly(A) mRNA isolation or our technical note for rRNA depletion.

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.

LIBRARY YIELDS

Figure 1. NEBNext Ultra II Directional RNA produces the highest yields, from a range of input amounts
Yield
Poly(A)-containing mRNA was isolated from Human Universal Reference RNA (Agilent #740000), and libraries were made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Kapa Stranded mRNA-Seq Kit, Kapa mRNA HyperPrep Kit and Illumina TruSeq Stranded mRNA Kit. The input RNA amount and number of PCR cycles are indicated. Library yields from an average of three replicates are shown.

View additional data on library yields.



GC CONTENT DISTRIBUTION

Figure 2. NEBNext Ultra II Directional RNA libraries provide uniform GC content distribution, at a broad range of input amounts
CG Plot
Poly(A)-containing mRNA was isolated from Human Universal Reference RNA (Agilent #740000), and libraries were made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Illumina TruSeq Stranded mRNA Kit, Kapa Stranded mRNA-Seq Kit and Kapa mRNA HyperPrep Kit. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Reads were mapped to the hg19 reference genome. GC content distribution for each library was calculated using mapped reads. Ultra II Directional RNA libraries had uniform GC content distribution across a range of input amounts, whereas for other kits the GC content distribution changed with different input amounts, indicating the introduction of input-dependent sequence bias.

View additional data on library quality.



MAXIMIZING TRANSCRIPT COVERAGE

Figure 3. NEBNext Ultra II Directional RNA libraries provide uniform coverage across the gene body of transcripts
coverage
Poly(A)-containing mRNA was isolated from Human Universal Reference RNA (Agilent #740000), and libraries were made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Illumina TruSeq Stranded mRNA Kit, Kapa Stranded mRNA-Seq Kit and Kapa mRNA HyperPrep Kit. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). This view of the 5´ to 3´coverage of RefSeq transcripts reveals consistent coverage for Ultra II Directional RNA libraries as input RNA is decreased from 1 μg to 10 ng. The changes apparent in other kits result from loss of coverage at the 3´ end of some transcripts.

View additional data on transcript coverage.



SUPERIOR LIBRARY COMPLEXITY AT LOW INPUT AMOUNTS

Figure 4. Low input NEBNext Ultra II Directional RNA libraries retain superior complexity
Transcription
Poly(A)-containing mRNA was isolated from Human Universal Reference RNA (Agilent #740000), and libraries were made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Illumina TruSeq Stranded mRNA Kit, Kapa Stranded mRNA-Seq Kit and Kapa mRNA HyperPrep Kit. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Salmon 0.4.0 was used for read mapping and quantification of all GENCODE v25 transcripts. TPM = Transcripts Per Kilobase Million. R2 values for the linear fit are shown. Correlation analysis of the transcripts indicates superior transcript expression correlation between the different inputs for Ultra II Directional RNA libraries.
View additional data on library complexity.


SUPERIOR PERFORMANCE WITH FFPE RNA

Figure 5. NEBNext Ultra II Directional RNA with NEBNext rRNA Depletion results in the lowest remaining ribosomal RNA levels with FFPE samples
FFPE low
Ribosomal RNA was depleted from human adult normal liver tissue FFPE Total RNA (Biochain # R2234149. RIN 2.5) and libraries were made using NEBNext Ultra II Directional RNA Kit (plus the NEBNext rRNA Depletion Kit (Human/Mouse/Rat)), Kapa Stranded RNA-Seq Kit with RiboErase, Kapa HyperPrep Kit with RiboErase, and Illumina TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero™ Gold. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Read pairs were assessed to be rRNA if they contain 6 or more 32 base matches to 18S, 28S, 5S, 5.8S, 16S or 12S human rRNA sequences (mirabait 4.9). Percent rRNA remaining was calculated by dividing rRNA reads by the total number of reads passing instrument quality filtering. Average percent rRNA remaining is shown for three replicates. The NEBNext rRNA Depletion Ultra II Directional RNA workflow is the most efficient in removing rRNA from total FFPE RNA.


Figure 6. Uniformity of Coverage across the AP000769.1-201 transcript
FFPR ERRC
Ribosomal RNA was depleted from human adult normal liver tissue FFPE Total RNA (Biochain # R2234149. RIN 2.5), and libraries were made using NEBNext Ultra II Directional RNA Kit (plus the NEBNext rRNA Depletion Kit (Human/Mouse/Rat)), Illumina TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero™ Gold, Kapa Stranded RNA-Seq Kit with RiboErase and Kapa HyperPrep Kit with RiboErase. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Coverage across the length of this individual transcript (ENST00000625158.1; AP000769.1-201) was assessed by mapping reads directly to the GENCODE v25 transcripts and examining 100 bins along the transcript length. NEBNext Ultra II Directional RNA libraries provided coverage across the entire length of the transcript even as input was decreased from 100 ng to 10 ng.

View additional data on FFPE RNA samples.

This product is related to the following categories:
Automation for NEBNext® NGS Library Prep,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation,
This product can be used in the following applications:
Applications of USER® and Thermolabile USER II Enzymes

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7760S     -20    
      NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext First Strand Synthesis Reaction Buffer E7421AVIAL -20 1 x 0.192 ml Not Applicable
      Random Primers E7422AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext Second Strand Synthesis Enzyme Mix E7425AVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix E7426AVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext USER® Enzyme E7428AVIAL -20 1 x 0.072 ml Not Applicable
      NEBNext® Ultra II End Prep Enzyme Mix E7646AVIAL -20 1 x 0.072 ml Not Applicable
      NEBNext® Ultra II End Prep Reaction Buffer E7647AVIAL -20 1 x 0.168 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml Not Applicable
      NEBNext® Ultra II Q5® Master Mix E7649AVIAL -20 1 x 0.6 ml Not Applicable
      NEBNext First Strand Synthesis Enzyme Mix E7761AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Adaptor Dilution Buffer E7762AVIAL -20 1 x 2.4 ml Not Applicable
      (0.1X) TE Buffer E7763AVIAL -20 1 x 2.78 ml Not Applicable
      Nuclease-free Water E7764AVIAL -20 1 x 1.25 ml Not Applicable
      NEBNext Strand Specificity Reagent E7766AVIAL -20 1 x 0.192 ml Not Applicable
  • E7760L     -20    
      NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext First Strand Synthesis Reaction Buffer E7421AAVIAL -20 1 x 0.768 ml Not Applicable
      Random Primers E7422AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext Second Strand Synthesis Enzyme Mix E7425AAVIAL -20 1 x 0.384 ml Not Applicable
      NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix E7426AAVIAL -20 1 x 0.768 ml Not Applicable
      NEBNext USER® Enzyme E7428AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Ultra II End Prep Enzyme Mix E7646AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Ultra II End Prep Reaction Buffer E7647AAVIAL -20 1 x 0.672 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml Not Applicable
      NEBNext® Ultra II Q5® Master Mix E7649AAVIAL -20 2 x 1.2 ml Not Applicable
      NEBNext First Strand Synthesis Enzyme Mix E7761AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Adaptor Dilution Buffer E7762AAVIAL -20 1 x 9.6 ml Not Applicable
      (0.1X) TE Buffer E7763AAVIAL -20 1 x 11.5 ml Not Applicable
      Nuclease-free Water E7764AAVIAL -20 1 x 5.7 ml Not Applicable
      NEBNext Strand Specificity Reagent E7766AAVIAL -20 1 x 0.768 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • NEBNext Singleplex or Multiplex Oligos for Illumina (NEB.com/oligos) or customer supplied oligos
  • Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)
  • SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
  • 80% Ethanol (freshly prepared)
  • Thermal Cycler
  • NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) 
    or NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400 or NEB #E7405)
    or NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7750 or NEB #E7755)
    or NEBNext® rRNA Depletion Kit (Bacteria) (NEB #E7850 or NEB #E7860) 
    or NEBNext® RNA Depletion Core Reagent Set (NEB #E7865 or NEB #E7870)
    or NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310 or NEB #E6350)

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.

Application Notes

FAQs & Troubleshooting

FAQs

  1. I have USER enzyme from my library prep kit (blue cap) and USER enzyme from my NEBNext Index Primer kit (red cap). Are they the same?
  2. What is the difference between the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) and the NEBNext Ultra II RNA Library Prep Kit (NEB #E7770)?
  3. What is the difference between the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) and the original NEBNext Ultra Directional RNA Library Prep Kit (NEB #E7420)?
  4. What is the starting material I need to use when preparing libraries using the NEBNext Ultra II Directional RNA kit?
  5. Which kit can I use to isolate Poly (A) mRNA from Total RNA?
  6. Which kit can I use to deplete ribosomal RNA (rRNA) from human, mouse or rat RNA?
  7. Does the kit include adaptor and primers?
  8. Is it possible to reduce the volume of beads added during the second strand synthesis cleanup step of the protocol for E7760, E7765, E7770, and E7775?
  9. Which NEBNext Oligos can be used with this library prep kit?
  10. Can I use this NEBNext kit with adaptors and primers from other vendors than NEB?
  11. What do I do if I see a precipitate in the Ultra II End Prep Reaction Buffer?

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.

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.

Licenses

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.

This product and its use are the subject of one or more issued and/or pending U.S. and foreign patent applications owned by Max Planck Gesellschaft. The purchase of this product from New England Biolabs, Inc., its affiliates, or its authorized resellers and distributors conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity) or for limited commercial purposes that are pre-approved by New England Biolabs, Inc. The purchase of this product does not convey a license under any claims in the foregoing patents or patent applications directed to producing the product. The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use this product for the following COMMERCIAL PURPOSES: (1) use of the products or its components in manufacturing; (2) use of the product or its components for therapeutic or prophylactic purposes in humans or animals.

For additional information or to inquire about commercial use, please contact busdev@neb.com.

Limited Use Label License: This product and its use are the subject of one or more issued and/or pending U.S. and foreign patent applications owned by Max Planck Gesellschaft. The purchase of this product from New England Biolabs, Inc., its affiliates, or its authorized resellers and distributors conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity) or for limited commercial purposes that are pre-approved by New England Biolabs, Inc. The purchase of this product does not convey a license under any claims in the foregoing patents or patent applications directed to producing the product. The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use this product for the following COMMERCIAL PURPOSES: (1) use of the products or its components in manufacturing; (2) use of the product or its components for therapeutic or prophylactic purposes in humans or animals.

Trademarks

KAPA® is a registered trademark of Kapa Biosystems
ILLUMINA®, TRUSEQ®, NEXTSEQ® and RIBO-ZERO® are registered trademarks of Illumina, Inc.
AGILENT® is a registered trademark of Agilent Technologies
SPRISELECT® and AMPURE® are registered trademarks of Beckman Coulter, Inc.

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