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


    The NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® contains enzymes and buffers that are ideally suited for cDNA library preparation for next-generation sequencing. Each of these components must pass rigorous quality control standards and are lot controlled, both individually and as a set of reagents.

    Ultra Directional RNA Library Preparation for IlluminaUltra Directional RNA Library Preparation for Illumina

    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.

    Functional Validation
    Each set of reagents is functionally validated together through construction and sequencing of an RNA library on the Illumina sequencing platform.

    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 First Strand Synthesis Reaction Buffer-205X
    Random Primers-20
    ProtoScript® II Reverse Transcriptase-20200,000 units/ml
    Murine RNase Inhibitor-20
    NEBNext Second Strand Synthesis Enzyme Mix-20
    NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix-2010X
    NEBNext End Prep Enzyme Mix-20
    NEBNext End Repair Reaction Buffer-2010X
    Blunt/TA Ligase Master Mix-20
    Nuclease-free Water-20
    NEBNext USER Enzyme
    NEBNext® Q5® Hot Start HiFi PCR Master Mix-20

    Advantages and 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 and 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) Oligos for Illumina or customer supplied Oligos
    • Magnetic Rack (Alpaqua, cat #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



    1. 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)?
    2. What is the starting material I need to use when preparing libraries using the NEBNext Ultra Directional RNA kit?
    3. What protocol do I use if starting with previously isolated mRNA or ribosomal depleted RNA?
    4. Which kit can I use to isolate Poly (A) mRNA from Total RNA?
    5. Does the kit provide adaptor and primers?


    1. Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module, NEB #E7490
    2. Protocol for use with NEBNext rRNA Depletion Kit (Human/Mouse/Rat), NEB #E6310
    3. Protocol for use with Purified mRNA or Ribosome Depleted RNA (E7420)


    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Usage Guidelines & Tips

    NEB Publications

    • Huang, L., Jin, J., Deighan, P., Liner, E., McReynolds, L. and Lieberman, J. (2013). Efficient and specific gene knockdown by small interfering RNAs produced in bacteria Nat Biotechnol.. PubMedID: 23475073, DOI: 10.1038/nbt.2537

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.