Product Class: Kit

NEBNext UltraExpress® RNA Library Prep Kit

For use in conjunction with this kit, the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) now has an Express protocol option for substantial time savings and a more streamlined workflow.


Catalog #E3330

Product Introduction

The NEBNext UltraExpress RNA Library Prep Kit is the latest generation of NEBNext RNA library prep, and it has a fast, streamlined workflow. With a 3-hour library prep protocol, the kit enables creation of high-quality RNA directional libraries in a single day, in conjunction with mRNA enrichment or rRNA depletion kits. 

  • Fast workflow (3 hours)
  • Fewer steps and consumables 
  • Fewer cleanups 
  • Single protocol for all input amounts
  • High-quality directional libraries from a broad input range: 25 - 250 ng total RNA
  • Automation friendly

View or download extensive performance data below or in our Data Supplement.

Product Information

Description

View or download extensive performance data below and in our Data Supplement
 
This new kit meets the need for substantially faster, more streamlined RNA library prep workflows that deliver high-quality directional libraries from a variety of sample types in a single day from total RNA. 

The kit allows the use of a single protocol for all RNA inputs (25 -250 ng total RNA) and incorporates master mixed reagents, reduced incubation times and fewer cleanup steps, reducing total time and consumables used.  

  • 3-hour library prep enables single day total RNA to library, in conjunction with mRNA enrichment or rRNA depletion kits
  • Fewer cleanups and pipetting steps reduce hands-on time
  • A robust single protocol for all input amounts increases ease of use
  • Flexibility is enabled with simple guidelines for customized protocols, if desired
  • Automation-friendly, enabling scalability

Figure 1: NEBNext UltraExpress® RNA Library Prep Kit workflow


Worklow Image


Figure 2: The NEBNext UltraExpress® RNA Library Prep Kit produces high library yields across a range of inputs


Graph of library yields

A. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent®), using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were prepared using the NEBNext UltraExpress® RNA Library Prep Kit, Kapa mRNA HyperPrep® Kit or Illumina® Stranded mRNA Kit. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions (20X for 76–250ng, 100X for 25–75 ng) and PCR cycle numbers (listed in figure).

B. Ribosomal RNA (rRNA) was depleted from UHRR, and libraries were prepared using the UltraExpress RNA Library Prep Kit (preceded by the NEBNext rRNA Depletion Kit (Human/Mouse/Rat – NEB #E7400), Kapa HyperPrep Kit with RiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero® Plus. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 11 PCR cycles for all inputs, or with customized adaptor dilutions (20X for 76–250 ng, 100X for 25–75 ng) and PCR cycle numbers (listed in figure).

The total RNA input amount and number of PCR cycles are indicated. Library yields calculated from an average of three replicates are shown with error bars indicating the standard deviation between replicates.    




Figure 3: The NEBNext UltraExpress® RNA Library Prep Kit provides consistent transcript coverage


Graphs of coverage

A. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent®), using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were prepared using the NEBNext UltraExpress® RNA Library Prep Kit, Kapa mRNA HyperPrep® Kit or Illumina® Stranded mRNA Kit. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent), and libraries were made using the UltraExpress RNA Library Prep Kit, Kapa mRNA HyperPrep Kit and Illumina Stranded mRNA Kit. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 10 PCR cycles for 250 ng input, 100X adaptor dilution with 12 PCR cycles (75 ng) and 14 PCR cycles (25 ng)).

B. Ribosomal RNA (rRNA) was depleted from UHRR, and libraries were prepared using the UltraExpress RNA Library Prep Kit (preceded by the NEBNext rRNA Depletion Kit (Human/Mouse/Rat – NEB #E7400), KAPA HyperPrep Kit with RiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero® Plus. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycles (20X adaptor dilution with 9 PCR cycles for 250 ng input, 100X adaptor dilution with 11 PCR cycles (75 ng) and 13 PCR cycles (25 ng)).

Libraries were sequenced on an Illumina NextSeq® 500 (2 x 75 bases). 10 million reads were sampled and mapped to the hg38 reference genome using RNA STAR v2.7.8a and 5´ to 3´ Transcript coverage was calculated from the top 1,000 transcripts using the CollectRnaSeqMetrics (Picard) tool v2.18.2.2.





Figure 4: The NEBNext UltraExpress® RNA Library Prep Kit provides greater sensitivity of transcript detection

Sensitivity graphs

A. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent®), using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were prepared using the NEBNext UltraExpress® RNA Library Prep Kit, Kapa mRNA HyperPrep® Kit or Illumina® Stranded mRNA Kit. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent), and libraries were made using the NEBNext UltraExpress RNA Library Prep Kit, Kapa mRNA HyperPrep Kit and Illumina Stranded mRNA Kit. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 10 PCR cycles for 250 ng input, 100X adaptor dilution with 12 PCR cycles (75 ng) and 14 PCR cycles (25 ng)).

B. Ribosomal RNA (rRNA) was depleted from UHRR, and libraries were prepared using the UltraExpress RNA Library Prep Kit (preceded by the NEBNext rRNA Depletion Kit (Human/Mouse/Rat – NEB #E7400), Kapa HyperPrep Kit with RiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero® Plus. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 11 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 9 PCR cycles for 250 ng input, 100X adaptor dilution with 11 PCR cycles (75 ng) and 13 PCR cycles (25 ng)).

Libraries were sequenced on an Illumina NextSeq® 500 (2 x 75 bases). 10 million reads were sampled and average transcript abundance was assessed for transcripts detected with ≤ 10 reads and ≤ 500 reads, respectively. Error bars indicate standard deviation for 3 replicates. Salmon v1.5.1 was used for mapping and quantification of all gencode v38 transcripts and ERCCs.





Figure 5: The NEBNext UltraExpress® RNA Library Prep Kit provides excellent transcript correlation between inputs and replicates
 
Transcript Correlation Graph

A. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent®), using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were prepared using the NEBNext UltraExpress RNA Library Prep with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 10 PCR cycles for 250 ng input, 100X adaptor dilution with 12 PCR cycles (75 ng) and 14 PCR cycles (25 ng)).

B. Ribosomal RNA (rRNA) was depleted from UHRR using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat – NEB #E7400) and libraries. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 11 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 9 PCR cycles for 250 ng input, 100X adaptor dilution with 11 PCR cycles (75 ng) and 13 PCR cycles (25 ng)).

Libraries were sequenced on an Illumina NextSeq® 500 (2 x 75 bases). The data was sampled from 10 million reads and libraries were correlated for transcript expression levels across inputs using Salmon v1.5.1 quantification of all gencode v38 transcripts and ERCCs. Each data point represents a transcript, with the log10 abundance in Number of Reads with 250 ng total RNA input on the y-axis compared to a replicate at 250 ng followed by 75 and 25 ng on the x-axis.





Figure 6: The NEBNext UltraExpress® RNA Library Prep Kit provides excellent transcript correlation across sample types

Graph of Sample Type

RNA from human whole blood (HWB) was depleted of globin mRNA and rRNA (A) using the NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat – NEB #E7750) or rRNA was depleted from a pool of 20 different bacterial organisms (ATCC® #MSA-2002) (B) using the NEBNext rRNA Depletion Kit (Bacteria – NEB #E7850). Libraries were prepared using the NEBNext UltraExpress® RNA Library Prep Kit, with customized adaptor dilution and PCR cycles per input (20X adaptor dilution with 9 PCR cycles for 250 ng input, 100X adaptor dilution with 11 PCR cycles (75 ng) and 13 PCR cycles (25 ng)). Libraries were sequenced on an Illumina® NextSeq® 500 (2 x 75 bases). The data was sampled from 10 million reads and libraries were correlated for transcript expression levels across inputs using Salmon v1.5.1 quantification of all gencode v38 transcripts (HWB) and a composite genome (Bacterial). Each data point represents a transcript, with the log10 abundance in number of Reads with 250 ng total RNA input on the y-axis compared to a replicate at 250 ng followed by 75 and 25 ng on the x-axis.



This product is related to the following categories:
Library Preparation for Illumina Products,
RNA Library Prep for Illumina,
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
  • E3330S     -20    
      NEBNext UltraExpress® RNA Fragmentation Mix E3329AVIAL -20 1 x 0.096 ml 2 X
      NEBNext UltraExpress® First Strand Enzyme Mix E3331AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext UltraExpress® Strand Specificity Reagent E3332AVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext UltraExpress® Second Strand Master Mix E3333AVIAL -20 1 x 0.72 ml Not Applicable
      NEBNext UltraExpress® End Prep Enzyme Mix E3334AVIAL -20 1 x 0.036 ml Not Applicable
      NEBNext UltraExpress® End Prep Reaction Buffer E3335AVIAL -20 1 x 0.06 ml Not Applicable
      NEBNext UltraExpress® Ligation Master Mix E3336AVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext UltraExpress® USER Enzyme E3337AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® MSTC™ High Yield Master Mix E3338AVIAL -20 1 x 1.2 ml Not Applicable
      NEBNext® Adaptor Dilution Buffer E7762AVIAL -20 1 x 2.4 ml Not Applicable
      0.1X TE E3341AVIAL -20 1 x 2.4 ml Not Applicable
      Nuclease-free Water E3342AVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext® Bead Reconstitution Buffer E3339AVIAL -20 1 x 0.096 ml Not Applicable
  • E3330L     -20    
      NEBNext UltraExpress® RNA Fragmentation Mix E3329AAVIAL -20 1 x 0.384 ml 2 X
      NEBNext UltraExpress® First Strand Enzyme Mix E3331AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext UltraExpress® Strand Specificity Reagent E3332AAVIAL -20 1 x 0.384 ml Not Applicable
      NEBNext UltraExpress® Second Strand Master Mix E3333AAVIAL -20 3 x 0.96 ml Not Applicable
      NEBNext UltraExpress® End Prep Enzyme Mix E3334AAVIAL -20 1 x 0.144 ml Not Applicable
      NEBNext UltraExpress® End Prep Reaction Buffer E3335AAVIAL -20 1 x 0.24 ml Not Applicable
      NEBNext UltraExpress® Ligation Master Mix E3336AAVIAL -20 1 x 1.16 ml Not Applicable
      NEBNext UltraExpress® USER Enzyme E3337AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® MSTC™ High Yield Master Mix E3338AAVIAL -20 1 x 4.8 ml Not Applicable
      NEBNext® Adaptor Dilution Buffer E7762AAVIAL -20 1 x 9.6 ml Not Applicable
      0.1X TE E3341AAVIAL -20 1 x 9.6 ml Not Applicable
      Nuclease-free Water E3342AAVIAL -20 1 x 0.384 ml Not Applicable
      NEBNext® Bead Reconstitution Buffer E3339AAVIAL -20 1 x 3.84 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • NEBNext Multiplex Oligos for Illumina®
  • NEBNext Multiplex Oligos options can be found at www.neb.com/oligos. Alternatively, customer supplied adaptor and primers can be used; please see information in link: https://www.neb.com/faqs/2019/03/08/can-i-use-this-nebnext-kit-with-adaptors-and-primers-from-other-vendors-than-neb
  • SPRIselect™ Reagent Kit (Beckman Coulter®, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
  • Magnetic Rack (NEB S1515S, Alpaqua® cat. #A001322, or equivalent)
  • 80% Ethanol (freshly prepared)
  • Thermal cycler
  • DNase-, RNase-free PCR strip tubes, for example TempAssure® PCR flex-free 8-tube strips (USA Scientific® #1402-4708)
  • Bioanalyzer® or TapeStation® (Agilent® Technologies, Inc.) and associated reagents and consumables

 

For use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490):

  • 1.5 ml Microcentrifuge tube and NEB #S1506 Magnet stand or equivalent (for washing beads only)

 

For use with NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400) and other NEBNext RNA depletion kits that do not include beads (NEB #E7750, E7850, E7865):

  • Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987)

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using the NEBNext UltraExpress® RNA Library Prep Kit (NEB #E3330)?

Manuals

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

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. Is the NEBNext UltraExpress® RNA library prep kit a directional (stranded) library kit?
  2. What is the difference between the NEBNext UltraExpress® RNA Library Prep Kit and the NEBNext® Ultra™ II Directional RNA Library Prep Kit? How do I choose the right one?
  3. Is it possible to use total RNA input amounts outside the suggested range for the NEBNext UltraExpress® RNA library Kit?
  4. Does the NEBNext UltraExpress® RNA Library Prep Kit include poly(A) mRNA isolation or rRNA Depletion reagents?
  5. Do the NEBNext UltraExpress® Library Prep kits come with beads?
  6. Can I perform poly(A) mRNA isolation or rRNA depletion and then freeze my samples at -80°C before beginning library preparation with the NEBNext UltraExpress® RNA Library Prep Kit?
  7. Which NEBNext® Multiplex Oligos (Adaptors & Primers) can be used with the NEBNext UltraExpress Library Prep Kits?
  8. What should I do if I see adaptor dimer in my NEBNext UltraExpress® libraries?
  9. Can this kit be used with adaptors and primers from suppliers other than NEB?
  10. Are the USER enzyme included in the NEBNext UltraExpress® RNA Library Prep Kit (blue cap) and the USER enzyme included with NEBNext Index Primer sets (red cap) the same?
  11. What sample types and quality are compatible with the NEBNext UltraExpress® RNA Library Prep Kit?
  12. What do I do if I see a precipitate in the Ultra II End Prep Reaction Buffer?

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.

Licenses

This product and its use are the subject of one or more issued and/or pending U.S. and foreign patent applications owned by Max Planck Gesellschaft. The purchase of this product from New England Biolabs, Inc., its affiliates, or its authorized resellers and distributors conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity) or for limited commercial purposes that are pre-approved by New England Biolabs, Inc. The purchase of this product does not convey a license under any claims in the foregoing patents or patent applications directed to producing the product. The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use this product for the following COMMERCIAL PURPOSES: (1) use of the products or its components in manufacturing; (2) use of the product or its components for therapeutic or prophylactic purposes in humans or animals.

Other Products You May Be Interested In