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  • NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 2)


    The novel NEBNext® Small RNA workflow has been optimized to minimize adaptor-dimers while producing high-yield, high-diversity libraries. The NEBNext® Small RNA Library Prep Set for Illumina® (Set 2) includes adaptors and multiplex primers with 12 indices, to enable multiplexing. An additional 12 index primers for multiplexing are included in kit NEB #E7300.
    Small RNA Library Preparation Workflow for IlluminaSmall RNA Library Preparation Workflow for Illumina

    GenomeWeb article reviews NEBNext Small RNA Kits.

    Functional Validation

    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 3´ Ligation Reaction Buffer-202X
    NEBNext 3´ SR Adaptor for Illumina
    NEBNext 5´ SR Adaptor for Illumina-20
    NEBNext 5´ Ligation Reaction Buffer-20
    NEBNext 5´ Ligation Enzyme Mix-20
    NEBNext SR RT Primer for Illumina
    NEBNext First Strand Synthesis Reaction Buffer-205X
    ProtoScript II Reverse Transcriptase-20200,000 units/ml
    Murine RNase Inhibitor
    LongAmp™ Taq 2X Master Mix-202X
    NEBNext SR Primer for Illumina-20
    Gel Loading Dye, Blue256X
    Quick-Load pBR322 DNA-MspI Digest-2050 μg/ml
    DNA Gel Elution Buffer-201X
    Linear Acrylamide-2010 mg/ml
    TE Buffer-20
    Nuclease-free Water-20
    NEBNext Index 13 Primer for Illumina
    NEBNext Index 14 Primer for Illumina
    NEBNext Index 15 Primer for Illumina
    NEBNext Index 16 Primer for Illumina
    NEBNext Index 17 Primer for Illumina
    NEBNext Index 18 Primer for Illumina
    NEBNext Index 19 Primer for Illumina
    NEBNext Index 20 Primer for Illumina
    NEBNext Index 21 Primer for Illumina
    NEBNext Index 22 Primer for Illumina
    NEBNext Index 23 Primer for Illumina
    NEBNext Index 24 Primer for Illumina
    NEBNext 3´ Ligation Enzyme Mix-20

    Properties and Usage

    Materials Required but not Supplied

    • 3 M Sodium Acetate, pH 5.5
    • 100% Ethanol
    • 80% Ethanol
    • Corning®, Costar®, Spin-X® Centrifuge Tube Filters (Cellulose Acetate Filters) (Sigma Aldrich # CLS8162)
    • Size Selection Materials: for gel size selection: [6% Novex® TBE PAGE gel,1.0 mM, 10-well (Life Technologies, Inc. #EC6265BOX), SYBR® Gold Nucleic Acid Gel Stain (Life Technologies, Inc. #S-11494)], RNase-free Disposable Pellet Pestles® (Kimble Kontes Asset Management, Inc. #749521-1590) or for bead selection: AMPure® XP Beads (Agencourt)
    • QIAquick® PCR Purification Kit (Qiagen #28104)
    • Dry Ice/Methanol Bath or -80°C freezer
    • Bioanalyzer® (Agilent Technologies, Inc.)

    Storage Temperature



    1. Can I use Total RNA to make small RNA libraries or do I have to isolate or enrich the sample for small RNA?
    2. Why does the RT primer hybridization occur before 5’adaptor ligation?
    3. Do I have to hybridize the RT primer again after 5’ ligation?
    4. How are the barcodes introduced in the Multiplex libraries?
    5. Are libraries prepared by this method compatible with paired-end flowcells for cluster generation?
    6. During size selection on 6% PAGE gel, which bands should I cut out of the gel?
    7. Can I use the small RNA sample preparation kit for Directional-RNA sequencing?


    1. Library Preparation (E7580)
    2. Validate the Library (For use with NEB #E7300, #E7330, and #E7580)
    3. QC Check and Size Selection using 6% PolyAcrylamide Gel (E7300)
    4. QC Check and Size Selection using AMPure XP Beads (E7300)
    5. QC Check and Size Selection Using Pippin Prep (E7300)


    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].

    Interactive Tools

    Application Notes


    • Macchiaroli, N. et al. (2015). microRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach. Parasites & Vectors. 8, 83. PubMedID: 25656283
    • Tsai, H.C. et al (2015). Transcriptional Analysis of Deinococcus radiodurans Reveals Novel Small RNAs That Are Differentially Expressed under Ionizing Radiation. Appl. Environ. Microbiol.. 81, 1754-1764. PubMedID: 25548054
    • Huang, X. et al. (2013). Characterization of human plasma-derived exosomal RNAs by deep sequencing BMC Genomics. 14, 319. PubMedID: 23663360, DOI: doi: 10.1186/1471-2164-14-319

    Safety Data Sheet

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