Product Class: Other

NEBNext® Multiplex Small RNA Library Prep Kit for Illumina® (Index Primers 1-48)

Product Introduction

The unique workflow of the NEBNext® Small RNA library prep kits addresses the challenge of minimization of adaptor-dimers while achieving production of high-yield, diverse multiplex libraries in a simple protocol.

  • Minimized adaptor-dimer contamination
  • High yields
  • Input RNA can be Total RNA
  • Suitable for methylated small RNAs (e.g., piRNAs) as well as unmethylated small RNAs
Catalog # Size Concentration
E7560S 96.0 reactions

Product Information

Description

The NEBNext Multiplex Small RNA Library Prep Kit for Illumina (Index Primers 1-48) contains the adaptors, primers, enzymes and buffers required to convert small RNAs into indexed libraries for next-generation sequencing on the Illumina platform. The novel workflow has been optimized to minimize adaptor-dimers, while producing high-yield, high-diversity libraries.

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

 

NEBNext Multiplex Small RNA Library Prep Kit for Illumina (Index Primers 1-48)

E7560 workflow figure
This product is related to the following categories:
Small RNA Library Preparation Products,
Next Generation Sequencing Library Preparation Products,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7560S     -20    
      Gel Loading Dye, Blue E6138AAVIAL -20 1 x 1 ml 6 X
      NEBNext 3´ Ligation Enzyme Mix E7288AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext Index 1 Primer for Illumina E7311AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 2 Primer for Illumina E7312AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 3 Primer for Illumina E7313AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 4 Primer for Illumina E7314AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 5 Primer for Illumina E7315AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 7 Primer for Illumina E7317AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 8 Primer for Illumina E7318AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 9 Primer for Illumina E7319AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 10 Primer for Illumina E7320AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 11 Primer for Illumina E7321AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 12 Primer for Illumina E7322AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 6 Primer for Illumina E7316AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext® Index 25 Primer for Illumina® E7561AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext® Index 27 Primer for Illumina® E7562AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext 3´ Ligation Reaction Buffer E7301AAVIAL -20 1 x 0.96 ml Not Applicable
      NEBNext 5´ Ligation Reaction Buffer E7304AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext 5´ Ligation Enzyme Mix E7305AAVIAL -20 1 x 0.24 ml Not Applicable
      Murine RNase Inhibitor E7308AAVIAL -20 1 x 0.096 ml Not Applicable
      LongAmp™ Taq 2X Master Mix E7309AAVIAL -20 4 x 1.2 ml Not Applicable
      NEBNext SR Primer for Illumina E7310AAVIAL -20 1 x 0.24 ml Not Applicable
      DNA Gel Elution Buffer E7324AAVIAL -20 1 x 48 ml Not Applicable
      Linear Acrylamide E7325AAVIAL -20 1 x 0.192 ml Not Applicable
      TE Buffer E7326AAVIAL -20 1 x 1.92 ml Not Applicable
      Nuclease-free Water E7327AAVIAL -20 1 x 20 ml Not Applicable
      NEBNext 5´ SR Adaptor for Illumina E7328AVIAL -20 1 x 1,350 pmol Not Applicable
      NEBNext 3´ SR Adaptor for Illumina E7332AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext SR RT Primer for Illumina E7333AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext First Strand Synthesis Reaction Buffer E7334AAVIAL -20 1 x 0.768 ml Not Applicable
      ProtoScript II Reverse Transcriptase E7355AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext Index 26 Primer for Illumina E7713AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 28 Primer for Illumina E7714AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 29 Primer for Illumina E7715AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 30 Primer for Illumina E7716AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 31 Primer for Illumina E7717AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 32 Primer for Illumina E7718AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 33 Primer for Illumina E7719AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 34 Primer for Illumina E7720AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 35 Primer for Illumina E7721AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 36 Primer for Illumina E7722AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 37 Primer for Illumina E7731AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 38 Primer for Illumina E7732AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 39 Primer for Illumina E7733AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 40 Primer for Illumina E7734AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 41 Primer for Illumina E7735AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 42 Primer for Illumina E7736AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 43 Primer for Illumina E7737AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 44 Primer for Illumina E7738AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 45 Primer for Illumina E7739AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 46 Primer for Illumina E7740AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 47 Primer for Illumina E7741AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 48 Primer for Illumina E7742AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 16 Primer for Illumina E7584AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 17 Primer for Illumina E7585AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 24 Primer for Illumina E7592AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 13 Primer for Illumina E7581AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 14 Primer for Illumina E7582AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 15 Primer for Illumina E7583AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 19 Primer for Illumina E7587AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 20 Primer for Illumina E7588AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 21 Primer for Illumina E7589AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 22 Primer for Illumina E7590AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 23 Primer for Illumina E7591AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext Index 18 Primer for Illumina E7586AVIAL -20 1 x 0.01 ml 10 µM
      Quick-Load pBR322 DNA-MspI Digest E7323AAVIAL -20 1 x 0.96 ml Not Applicable

Properties & 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)
Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)

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.)

Protocols, Manuals & Usage

Protocols

  1. Protocol for use with NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)
  2. 6A. Quality Control Check and Size Selection using 6% Polyacrylamide Gel  NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)
  3. 6B. Quality Control Check and Size Selection using Pippin Prep NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)
  4. 6C. Quality Control Check and Size Selection using AMPure XP Beads NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)

Manuals

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

FAQs & Troubleshooting

FAQs

  1. Is it possible to prevent abundant, unwanted RNAs to be included in the Small RNA library?
  2. Can I use Total RNA to make small RNA libraries or do I have to isolate or enrich the sample for small RNA?
  3. Why does the RT primer hybridization occur before 5’adaptor ligation?
  4. Do I have to hybridize the RT primer again after 5’ ligation?
  5. How are the barcodes introduced in the Multiplex libraries?
  6. During size selection on 6% PAGE gel, which bands should I cut out of the gel?
  7. Are libraries prepared with the NEBNext® Small RNA Library prep kit for Illumina compatible with paired-end flow cells for cluster generation?
  8. Can I use the small RNA sample preparation kit for Directional-RNA sequencing?
  9. What is the sequence of the final PCR product?
  10. Can I use enriched small RNA instead of total RNA for the NEBNext Small RNA Library Prep for Illumina?
  11. What should I do if my Small RNA may not have 5´ Phosphate and 3´ OH groups?
  12. How should my NEBNext Small RNA library be trimmed?
  13. What should I do if the sample sheet from the NEB.com website is not compatible with the software version I am using?
  14. Do you have Sample Sheets for use with the NEBNext Multiplex Small RNA Library Prep for Illumina?
  15. Are NEBNext adaptor and primers for Illumina compatible with NEBNext reagents for Illumina library preparation?
  16. Do I have to use custom sequencing primers for my libraries generated with NEBNext® Small RNA Library prep kit for Illumina?

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

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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