Product Class: Kit

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

Note that additional index primers for multiplexing are provided with kits NEB #E7300 and NEB #E7560.

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
E7580S 24.0 reactions
E7580L 96.0 reactions

Product Information

Description



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. Additional index primers for multiplexing are included in kits NEB #E7300 and NEB #E7560.

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.

Small RNA Library Preparation Workflow for Illumina



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

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7580S     -20    
      NEBNext 3´ Ligation Reaction Buffer E7301AVIAL -20 1 x 0.24 ml Not Applicable
      NEBNext 3´ SR Adaptor for Illumina E7332AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext 5´ SR Adaptor for Illumina E7328AVIAL -20 1 x 1,350 pmol Not Applicable
      NEBNext 5´ Ligation Reaction Buffer E7304AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext 5´ Ligation Enzyme Mix E7305AVIAL -20 1 x 0.06 ml Not Applicable
      NEBNext SR RT Primer for Illumina E7333AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext First Strand Synthesis Reaction Buffer E7334AVIAL -20 1 x 0.192 ml Not Applicable
      ProtoScript II Reverse Transcriptase E7355AVIAL -20 1 x 0.024 ml Not Applicable
      Murine RNase Inhibitor E7308AVIAL -20 1 x 0.024 ml Not Applicable
      LongAmp™ Taq 2X Master Mix E7309AVIAL -20 1 x 1.2 ml Not Applicable
      NEBNext SR Primer for Illumina E7310AVIAL -20 1 x 0.06 ml Not Applicable
      Gel Loading Dye, Blue E6138AVIAL -20 1 x 0.2 ml 6 X
      Quick-Load pBR322 DNA-MspI Digest E7323AVIAL -20 1 x 0.24 ml Not Applicable
      DNA Gel Elution Buffer E7324AVIAL -20 1 x 12 ml Not Applicable
      Linear Acrylamide E7325AVIAL -20 1 x 0.048 ml Not Applicable
      TE Buffer E7326AVIAL -20 1 x 0.48 ml Not Applicable
      Nuclease-free Water E7327AVIAL -20 1 x 5 ml Not Applicable
      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 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 18 Primer for Illumina E7586AVIAL -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 24 Primer for Illumina E7592AVIAL -20 1 x 0.01 ml 10 µM
      NEBNext 3´ Ligation Enzyme Mix E7288AVIAL -20 1 x 0.072 ml Not Applicable
  • E7580L     -20    
      NEBNext 3´ Ligation Reaction Buffer E7301AAVIAL -20 1 x 0.96 ml Not Applicable
      NEBNext 3´ SR Adaptor for Illumina E7332AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext 5´ SR Adaptor for Illumina E7328AVIAL -20 1 x 1,350 pmol 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
      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
      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
      Gel Loading Dye, Blue E6138AAVIAL -20 1 x 1 ml 6 X
      Quick-Load pBR322 DNA-MspI Digest E7323AAVIAL -20 1 x 0.96 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 Index 13 Primer for Illumina E7581AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 14 Primer for Illumina E7582AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 15 Primer for Illumina E7583AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 16 Primer for Illumina E7584AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 17 Primer for Illumina E7585AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 18 Primer for Illumina E7586AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 19 Primer for Illumina E7587AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 20 Primer for Illumina E7588AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 21 Primer for Illumina E7589AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 22 Primer for Illumina E7590AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 23 Primer for Illumina E7591AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext Index 24 Primer for Illumina E7592AAVIAL -20 1 x 0.04 ml 10 µM
      NEBNext 3´ Ligation Enzyme Mix E7288AAVIAL -20 1 x 0.288 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)


Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB# T1030)
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.

Application Notes

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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