Product Class: Kit

NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina®
Item# Description List Price Web Price Qty
E7760L NEBNext Ultra II Directional RNA Library Prep Kit for Illumina - 96 rxns $4,604.00
$4,143.60
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E7760S NEBNext Ultra II Directional RNA Library Prep Kit for Illumina - 24 rxns $1,356.00
$1,220.40
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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.

    • Generate the highest yields of high quality libraries, with a broad range of input amounts:
      • 10 ng – 1 µg Total RNA (polyA mRNA workflow)
      • 5 ng – 1 µg Total RNA (rRNA depletion workflow)
    • Increase the complexity and transcript coverage of your libraries
    • Optimize your time with streamlined workflows, reduced hands-on time, and automation compatibility
    • Rely on robust performance, even with low quality RNA, including FFPE

    Product Information

    Description

    Ultra II Sample
     
     
    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 5 ng or 10 ng of Total RNA, respectively, up to 1µg.

    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
    • Get more of what you need, with the highest library yields
    • Generate high quality libraries even when you have only limited amounts of input RNA:
      • 10 ng – 1 µg Total RNA (polyA mRNA workflow)
      • 5 ng – 1 µg Total RNA (rRNA depletion workflow)
    • Minimize bias, with fewer PCR cycles required
    • Increase the complexity and transcript coverage of your libraries
    • Optimize your time with streamlined workflows, reduced hands-on time, and automation compatibility
    • Rely on robust performance, even with low quality RNA, including FFPE
    • Compatible with NEBNext poly(A) mRNA Isolation, rRNA Depletion reagents and multiplexing adaptors and primers.
    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.

    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.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    NEBNext® Ligation Enhancer -20
    NEBNext First Strand Synthesis Reaction Buffer -20
    Random Primers -20
    NEBNext Second Strand Synthesis Enzyme Mix -20
    NEBNext USER® Enzyme -20
    NEBNext Ultra II End Prep Enzyme Mix -20
    NEBNext Ultra II End Prep Reaction Buffer -20
    NEBNext® Ultra II Ligation Master Mix -20
    NEBNext® Ultra II Q5® Master Mix -20 2X
    NEBNext First Strand Synthesis Enzyme Mix -20
    NEBNext Adaptor Dilution Buffer -20 1X
    (0.1X) TE Buffer -20 0.1X
    Nuclease-free Water -20
    NEBNext Strand Specificity Reagent -20
    Product Categories:
    Sample Prep for NGS & Target Enrichment Products
    Applications:
    RNA Library Preparation,
    Applications of USER® and Thermolabile USER II Enzymes

    Properties & Usage

    Materials Required but not Supplied

    • NEBNext Singleplex (NEB #E7350) or NEBNext Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600) Oligos for Illumina or customer supplied oligos
    • Magnetic Rack (Alpaqua, cat. #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 (Human/Mouse/Rat) (NEB #E6310)

    Storage Temperature

    -20°C

    Protocols, Manuals & Usage

    Protocols

    1. Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765)
    2. Protocol for use with NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765)
    3. Protocol for use with FFPE RNA, NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765)
    4. Protocol for use with Purified mRNA or rRNA Depleted RNA and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765)
    5. Protocol for use with rRNA Depleted FFPE RNA and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765)
    6. Appendix A for use with Ultra™ II RNA and Ultra II Directional RNA (E7760, E7765, E7770, E7775)

    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?

    Troubleshooting

    • NEBNext® RNA Library Prep Kit Troubleshooting Guide (NEB #E7760, E7765, E7770, E7775, E7420, E7530)

    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

    This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

    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.

    For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

    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.

    Licenses

    This product is licensed 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 for use only in: (a) standard (not real-time) PCR in the research field only, but not real time PCR or digital PCR; (b) real-time PCR for use as a library preparation quantitation tool in Next Generation Sequencing workflows; (c) any in-vitro diagnostics applications, except for applications using real-time PCR or digital PCR; and (d) any non-PCR applications in DNA sequencing, isothermal amplification, and the production of synthetic DNA.

    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.