Product Class:

NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®
Item# Description List Price Web Price Qty
E7420L NEBNext Ultra Directional RNA Library Prep Kit for Illumina - 96 rxns $5,398.00
$4,858.20
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E7420S NEBNext Ultra Directional RNA Library Prep Kit for Illumina - 24 rxns $1,664.00
$1,497.60
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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

    Product Introduction

    This original version of the Ultra Directional RNA Library Prep Kit enables strand-specific RNA library preparation in a streamlined workflow. However, we now recommend the new NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760), which has been further optimized to provide increased yields of high quality libraries from a broader range of input amounts.

    Product Information

    Description




    The NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® contains reagents for preparation of strand-specific RNA libraries for Illumina sequencing. Please note that adaptors, primers, rRNA depletion reagents and poly(A) mRNA isolation reagents are not included in the kit and are available separately.

    Each kit component must pass rigorous quality control standards, and each set of reagents is functionally validated together by construction and sequencing of a library on the Illumina sequencing platform.

    For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact [email protected] for further information.


    Ultra Directional RNA Library Preparation for IlluminaUltra Direct

    NEBNext Ultra™ Directional RNA produces libraries with high directionality/strand specificityNEBNext Ultra™ Directional RNA produces libraries with high directionality/strand specificity

    Ultra Directional RNA libraries of mRNA purified from Universal Human Reference RNA (Agilent® #740000), were sequenced on the Illumina platform. The high percentage of reads mapped to the expected strand shows the high strand-specificity of the libraries. Actinomycin D is required to achieve maximal strand specificity.

    NEBNext Ultra™ Directional RNA libraries provide even coverage across transcriptsNEBNext Ultra™ Directional RNA libraries provide even coverage across transcripts

    mRNA was isolated from Universal Human Reference RNA (Agilent # 740000) and libraries were made using the NEBNext Ultra™ Directional RNA Kit and the Illumina TruSeq Stranded mRNA Kit. Libraries were sequenced (single end reads) on the Illumina platform (GAIIx). Good coverage across expressed transcripts was obtained.

    Low input NEBNext Ultra Directional RNA libraries retain complexityLow input NEBNext Ultra Directional RNA libraries retain complexity

    Ribosomal RNA-depleted RNA libraries were made from 10 ng or 100 ng Universal Human Reference RNA (Agilent #740000) using the NEBNext Ultra Directional RNA Kit. Transcript abundances in Fragments Per Kilobase of exon per Million fragments mapped (FPKM) show strong concordance (R2 = 0.90745), indicating retention of library complexity even at low input amount.

    Equivalent transcript representation is achieved between technical replicates of NEBNext Ultra Directional RNA librariesEquivalent transcript representation is achieved between technical replicates of NEBNext Ultra Directional RNA libraries

    Technical replicates of mRNA libraries made from mRNA isolated from Universal Human Reference RNA (Agilent #740000) show strong concordance (R2 = 0.99409).


    Sequencing data shows strong concordance between NEBNext Ultra Directional RNA and Illumina TruSeq Stranded RNA librariesSequencing data shows strong concordance between NEBNext Ultra Directional RNA and Illumina TruSeq Stranded RNA libraries

    mRNA was isolated from Universal Human Reference RNA (Agilent #740000) and libraries were made using the NEBNext Ultra Directional RNA Kit and the Illumina TruSeq Stranded mRNA Kit. This FPKM concordance plot shows comparable transcript representation (R2 = 0.96009) between the libraries.

    Each set of reagents is functionally validated together through construction and sequencing of a transcriptome library and sequenced on a

    Lot Control

    The lots provided are managed separately and qualified by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays, and also meet stringent criteria in the additional quality controls listed on each individual component page

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    NEBNext End Prep Enzyme Mix -20
    NEBNext End Repair Reaction Buffer -20 10X
    Blunt/TA Ligase Master Mix -20
    NEBNext First Strand Synthesis Reaction Buffer -20
    Random Primers -20
    ProtoScript® II Reverse Transcriptase -20 200,000 units/ml
    Murine RNase Inhibitor -20
    NEBNext Second Strand Synthesis Enzyme Mix -20
    NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix -20 10X
    NEBNext USER® Enzyme -20
    Nuclease-free Water -20
    NEBNext® Q5® Hot Start HiFi PCR Master Mix -20
    Product Categories:
    RNA Library Prep Products
    Applications:
    RNA-seq,
    RNA For Illumina,
    Illumina Library Preparation

    Advantages and Features

    Features

    • Accurate retention of transcript strand of origin information
    • Utilizes dUTP-based methodology
    • Low input amounts (as low as 10ng purified mRNA or ribosomal-depleted RNA, or 100ng Total RNA)
    • Fast workflow (4.5 – 5 hours), with minimal hands-on time
    • Robust, reliable performance
    • Ultra high fidelity amplification with minimized GC bias

    Properties & Usage

    Materials Required but not Supplied

    • NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or NEBNext rRNA Depletion Kit (NEB #E6310)
    • NEBNext Singleplex (NEB #E7350) or NEBNext Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E6609#E7600) Oligos for Illumina or customer supplied Oligos
    • Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)
    • 80% Ethanol (freshly prepared) 
    • 0.1X TE, pH 8.0
    • 10 mM Tris-HCl, pH 7.5–8.0
    • 10 mM NaCl (optional)
    • Agencourt® AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
    • Actinomycin D (Sigma# A1410, dissolved in dimethylsulfoxide [DMSO] to 5 μg/ul)
    • Additional required materials not included for use with NEBNext rRNA Depletion Kit (NEB #E6310): Agencourt RNAClean® XP (Beckman Coulter, Inc., Cat #A63987)

    Storage Temperature

    -20°C

    Protocols, Manuals & Usage

    Protocols

    1. Protocol for use with NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420)
    2. Protocol for use with Purified mRNA or Ribosome Depleted RNA - NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420)
    3. Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420)

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

    Usage & Guidelines

    • Getting Started with RNA-Sequencing (RNA-Seq)

    Faqs & Troubleshooting

    FAQs

    1. Is the Monarch® Total RNA Miniprep Kit compatible with NEBNext® reagents for RNA Library Prep?
    2. What is the difference between the NEBNext Ultra Directional RNA library prep kit for Illumina (E7420) and the NEBNext Ultra RNA library prep kit for Illumina (E7530)?
    3. What is the starting material I need to use when preparing libraries using the NEBNext Ultra Directional RNA kit?
    4. What protocol do I use if starting with previously isolated mRNA or ribosomal depleted RNA?
    5. Which kit can I use to isolate Poly (A) mRNA from Total RNA?
    6. Does the kit provide adaptor and primers?
    7. How do I know the concentration of my index primers? 
    8. I have USER® Enzyme from my NEBNext® library prep kit (blue cap) and USER Enzyme from my NEBNext Index Primer kit (red cap). Are they the same?
    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?

    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 covered by one or more Patents. 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.