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  • NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1)


    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 1) includes adaptors and multiplex primers with 12 indices, to enable multiplexing. An additional 12 index primers for multiplexing are included in kit NEB #E7580.

    Small RNA Library Preparation Workflow for Illumina

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

    GenomeWeb article reviews NEBNext Small RNA Kits.

    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 5´ SR Adaptor for Illumina-20
    NEBNext 5´ Ligation Reaction Buffer-20
    Murine RNase Inhibitor
    LongAmp™ Taq 2X Master Mix-202X
    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 1 Primer for Illumina-20
    NEBNext Index 2 Primer for Illumina-20
    NEBNext Index 3 Primer for Illumina-20
    NEBNext Index 4 Primer for Illumina-20
    NEBNext Index 5 Primer for Illumina-20
    NEBNext Index 6 Primer for Illumina-20
    NEBNext Index 7 Primer for Illumina-20
    NEBNext Index 8 Primer for Illumina-20
    NEBNext Index 9 Primer for Illumina-20
    NEBNext Index 10 Primer for Illumina-20
    NEBNext Index 11 Primer for Illumina-20
    NEBNext Index 12 Primer for Illumina-20
    NEBNext 3´ Ligation Enzyme Mix-20
    NEBNext 5´ Ligation Enzyme Mix-20
    NEBNext 3´ SR Adaptor for Illumina
    ProtoScript II Reverse Transcriptase-20200,000 units/ml
    NEBNext First Strand Synthesis Reaction Buffer-205X
    NEBNext SR RT Primer for Illumina
    NEBNext SR Primer for Illumina-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)
    Dry Ice/Methanol Bath or –80°C freezer

    for bead selection:
    Agencourt® AMPure® XP Beads (Beckman Coulter, Inc. #A63881)

    for Pippin Prep™ selection:
    3% Agarose Dye Free Gel (Sage Science #CDF 3010)

    QIAquick PCR Purification Kit (Qiagen® #28104)
    Bioanalyzer® (Agilent® Technologies, Inc.)

    Storage Temperature



    1. Kit Optimization

      The components of this kit were collectively optimized with FirstChoice® Human Brain Reference RNA (Life Technologies, Inc. #AM6050).

      High quality total RNA should be used as starting material. An RNA Integrity Number (RIN) > 7 is recommended.

      Assess the quality and quantity of your sample using the Agilent 2100 Bioanalyzer using an Agilent RNA 6000 Nano Chip.


    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. During size selection on 6% PAGE gel, which bands should I cut out of the gel?
    6. Are libraries prepared by this method compatible with paired-end flowcells for cluster generation?
    7. Can I use the small RNA sample preparation kit for Directional-RNA sequencing?
    8. What is the sequence of the final PCR product?


    1. Library Preparation (E7300)
    2. QC Check and Size Selection using AMPure XP Beads (E7300)
    3. QC Check and Size Selection using 6% PolyAcrylamide Gel (E7300)
    4. QC Check and Size Selection Using Pippin Prep (E7300)
    5. Validate the Library
    6. Size Selection using AMPure XP Beads


    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].

    Application Notes


    • 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
    • Macchiaroli, N. et al. (2015)microRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach. Parasites & Vectors 8, 83. PubMedID: 25656283
    • 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.