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


    The NEBNext® Ultra™ 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.

    NEBNext Ultra™ RNA provides even sequence coverage across transcripts for a wide range of inputsNEBNext Ultra™ RNA provides even sequence coverage across transcripts for a wide range of inputs

    10 ng, 100ng or 1 μg of Total RNA (Universal Human Reference RNA, Agilent #740000) was used as input for libraries, following the NEBNext Ultra™ RNA protocol and using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were sequenced (single end reads) on the Illumina platform (GAIIx). Good coverage across expressed transcript was obtained for each input amount.

    Ultra Non-directional RNA Library Preparation for IlluminaUltra Non-directional RNA Library Preparation for Illumina

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

    Advantages and Features


    • Low input amounts (as low as 10ng total RNA, purified mRNA or ribosomal-depleted RNA)
    • Fast workflow (5 – 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 (Human/Mouse/Rat) (NEB #E6310)
    • NEBNext Singleplex (NEB #E7350) or NEBNext Multiplex (NEB #E7335, #E7500 or #7600) Oligos for Illumina
    • Magnetic Rack (Alpaqua, cat #A001322 or equivalent)
    • 80% Ethanol (freshly prepared)
    • Agencourt® AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
    • Agencourt RNAClean® XP (Beckman Coulter, Inc. #A63987) are required when using NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310)
    • 0.1X TE, pH 8.0
    • 10mM Tris-HCl, pH 7.5–8.0
    • 10 mM NaCl (optional)

    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. Where do I have to start the protocol if I have purified 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?
    6. I have an index primer kit PN E7335, E7500 or E7535 with a lot number not listed in the manual. Is the concentration of the primers 10 µM or 25 µM? 


    1. Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module, NEB #E7490 and NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #E7530)
    2. Protocol for use with NEBNext rRNA Depletion Kit (Human/Mouse/Rat), NEB #E6310 and NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #E7530)
    3. Protocol for use with Purified mRNA or Ribosome Depleted RNA - NEBNext Ultra RNA Library Prep Kit o for Illumina (E7530)


    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

    Interactive Tools

    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.. 31(4), 350-356. PubMedID: 23475073, DOI: 10.1038/nbt.2537


    • Fustin, J.-M. et al. (2013). RNA-Methylation-Dependent RNA Processing Controls the Speed of the Circadian Clock Cell. 155(4), 793-806. PubMedID: 24209618, DOI: 10.1016/j.cell.2013.10.026

    Safety Data Sheet

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