Product Class: Kit

NEBNext® Ultra™ II RNA Library Prep with Sample Purification Beads

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.

Product Introduction

Do you prefer to use non-strand-specific library prep methods, but need increased sensitivity and specificity from your RNA-seq experiments, from ever-decreasing amounts of input RNA? To address these challenges, our next generation of non-directional RNA library prep kit has been reformulated at each step, resulting in several fold higher yields of high quality libraries and enabling use of lower input amounts and fewer PCR cycles.

  • Generate the highest yields of high quality libraries, with a broad range of input amounts

10 ng – 1 µg Total RNA (polyA mRNA workflow)

10 ng – 1 µg Total RNA (rRNA depletion workflow)

  • Increase the complexity and transcript coverage of your libraries
  • Optimize your time with streamlined workflows, reduced hands-on time, and automation compatibility
  • Enjoy the flexibility and reliability of the gold standard SPRIselect size selection and clean-up beads, supplied in just the amounts you need

 

Catalog # Size Concentration
E7775S 24.0 reactions
E7775L 96.0 reactions

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Product Information

Description

Ultra II Sample
 

The Ultra II RNA Library Prep Kit for Illumina is designed for non-directional (non-strand-specific) RNA library construction, and delivers significantly increased sensitivity and specificity from your RNA-seq experiments, from ever-decreasing amounts of input RNA. In conjunction with ribosomal RNA (rRNA) depletion or poly(A) mRNA enrichment, the kit enables the production of high quality libraries from 10 ng of Total RNA, respectively, up to 1µg.

For directional (strand-specific) RNA library preparation, see the NEBNext Ultra II Directional RNA Library Prep kit for Illumina.

See what customers are saying about NEBNext Ultra II RNA.

Features

Also available without optional SPRIselect® beads.

For extensive NEBNext Ultra II performance data, click the links in the Features above and download our technical note for poly(A) mRNA isolation or our technical note for rRNA depletion.


LIBRARY YIELDS

Figure 1. NEBNext Ultra II RNA produces the highest yields, from a range of input amounts
Yield
Poly(A)-containing mRNA was isolated from Human Universal Reference RNA (Agilent #740000) and libraries were prepared using the NEBNext Ultra II RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), and the Illumina TruSeq RNA Sample Preparation Kit v2. Input RNA and number of PCR cycles are indicated. Library yields from an average of three replicates are shown.

View additional data on library yields


DUPLICATION RATES

Figure 2. NEBNext Ultra II RNA with NEBNext rRNA Depletion results in lower duplication rates .
Duplication
Ribosomal RNA was depleted from 1 μg, 100 ng and 5 ng of Human Universal Reference RNA (Agilent #740000) using the NEBNext rRNA Depletion Kit (Human/ Mouse/Rat) or Illumina Ribo-Zero™ Gold rRNA Removal Kit (Human/Mouse/Rat). Libraries were then prepared using the NEBNext Ultra II RNA Kit or the Illumina TruSeq RNA Library Prep Kit v2, respectively. 5 ng input was tested only with the NEBNext kits. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Reads were down sampled to 10 million read pairs and mapped to the hg19 reference genome. Duplication rates were calculated as a fraction of uniquely mapped reads using the ‘Read Duplication’ tool of RSeQC where reads mapping to the same genomic location are regarded as duplicated reads.

View additional data on library quality


MAXIMIZING TRANSCRIPT COVERAGE

Figure 3. NEBNext Ultra II RNA libraries provide uniform coverage across the gene body of transcripts
Coverage
Poly(A)-containing mRNA was isolated from Human Universal Reference RNA (Agilent #740000), and libraries were prepared using the NEBNext Ultra II RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), and the Illumina TruSeq RNA Sample Preparation Kit v2. Input RNA amount is indicated. Libraries were sequenced on an Illumina® NextSeq® 500 using paired-end mode (2x76 bp). This view of the 5´ to 3´ coverage of RefSeq transcripts reveals consistent coverage for Ultra II RNA libraries as input RNA is decreased from 1 μg to 10 ng. The changes apparent in The TruSeq kit results from loss of coverage at the 3´ end of some transcripts.

View additional data on transcript coverage


EXCELLENT LIBRARY COMPLEXITY AT LOW INPUT AMOUNTS

Figure 4. Low input NEBNext Ultra II RNA libraries retain complexity even at low input amounts
Complexity
Ribosomal RNA was depleted from 1 μg, 100 ng and 5 ng of Human Universal Reference RNA (Agilent #740000) with recommended amounts of ERCC RNA Spike-In Mix I (Thermo Fisher Scientific #4456740) using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and libraries were then prepared using the NEBNext Ultra II RNA Kit. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Salmon 0.4.0 was used for read mapping and quantification of all ERCC transcripts. R2 values for linear fit are shown. TPM (Transcripts Per Kilobase Million) correlation analysis of the transcripts indicates excellent transcript expression correlation between the different inputs for Ultra II RNA libraries (A), including ERCC transcripts (B).

View additional data on library complexity
This product is related to the following categories:

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7775S     Multi-temperature    
      NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® E7770S -20 1 x 24 reactions Not Applicable
      NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext First Strand Synthesis Reaction Buffer E7421AVIAL -20 1 x 0.192 ml Not Applicable
      Random Primers E7422AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext Second Strand Synthesis Enzyme Mix E7425AVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext Second Strand Synthesis Reaction Buffer E7427AVIAL -20 1 x 0.192 ml Not Applicable
      Nuclease-free Water E7764AVIAL -20 1 x 1.25 ml Not Applicable
      NEBNext® Ultra II End Prep Enzyme Mix E7646AVIAL -20 1 x 0.072 ml Not Applicable
      NEBNext® Ultra II End Prep Reaction Buffer E7647AVIAL -20 1 x 0.168 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml Not Applicable
      NEBNext® Ultra II Q5® Master Mix E7649AVIAL -20 1 x 0.6 ml Not Applicable
      NEBNext First Strand Synthesis Enzyme Mix E7761AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Adaptor Dilution Buffer E7762AVIAL -20 1 x 2.4 ml Not Applicable
      (0.1X) TE Buffer E7763AVIAL -20 1 x 2.78 ml Not Applicable
      NEBNext® Sample Purification Beads E7767S 25 1 x 6.65 ml Not Applicable
      NEBNext® Sample Purification Beads E7767SVIAL 25 1 x 6.65 ml Not Applicable
  • E7775L     Multi-temperature    
      NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® E7770L -20 1 x 96 reactions Not Applicable
      NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext First Strand Synthesis Reaction Buffer E7421AAVIAL -20 1 x 0.768 ml Not Applicable
      Random Primers E7422AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext Second Strand Synthesis Enzyme Mix E7425AAVIAL -20 1 x 0.384 ml Not Applicable
      NEBNext Second Strand Synthesis Reaction Buffer E7427AAVIAL -20 1 x 0.768 ml Not Applicable
      Nuclease-free Water E7764AAVIAL -20 1 x 5.7 ml Not Applicable
      NEBNext® Ultra II End Prep Enzyme Mix E7646AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Ultra II End Prep Reaction Buffer E7647AAVIAL -20 1 x 0.672 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml Not Applicable
      NEBNext® Ultra II Q5® Master Mix E7649AAVIAL -20 2 x 1.2 ml Not Applicable
      NEBNext First Strand Synthesis Enzyme Mix E7761AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Adaptor Dilution Buffer E7762AAVIAL -20 1 x 9.6 ml Not Applicable
      (0.1X) TE Buffer E7763AAVIAL -20 1 x 11.5 ml Not Applicable
      NEBNext® Sample Purification Beads E7767L 25 1 x 26.6 ml Not Applicable
      NEBNext® Sample Purification Beads E7767SVIAL 25 4 x 6.65 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • NEBNext Singleplex or Multiplex Oligos for Illumina (NEB.com/oligos) or customer supplied oligos
  • Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)
  • 80% Ethanol (freshly prepared)
  • Thermal Cycler
  • NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) 
    or NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400 or NEB #E7405)
    or NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7750 or NEB #E7755)
    or NEBNext® rRNA Depletion Kit (Bacteria) (NEB #E7850 or NEB #E7860) 
    or NEBNext® RNA Depletion Core Reagent Set (NEB #E7865 or NEB #E7870)
    or NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310 or NEB #E6350)

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using NEBNext® RNA Library Prep reagents?

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. What is the difference between the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (NEB #E7775) and the NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads (NEB #E7765)?
  2. What is the starting material I need to use when preparing libraries using the NEBNext Ultra II Directional RNA kit?
  3. Which kit can I use to isolate Poly (A) mRNA from Total RNA?
  4. Which kit can I use to deplete ribosomal RNA (rRNA) from human, mouse or rat RNA?
  5. Does the kit include adaptor and primers?
  6. Is it possible to reduce the volume of beads added during the second strand synthesis cleanup step of the protocol for E7760, E7765, E7770, and E7775?
  7. Which NEBNext Oligos can be used with this library prep kit?
  8. Can I use this NEBNext kit with adaptors and primers from other vendors than NEB?
  9. What do I do if I see a precipitate in the Ultra II End Prep Reaction Buffer?
  10. Is it possible to reduce the volume of beads added during the second strand synthesis cleanup step?

Troubleshooting

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]

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 is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.

For additional information or to inquire about commercial use, please contact busdev@neb.com.

Trademarks

ILLUMINA®, TRUSEQ®, NEXTSEQ® and RIBO-ZERO® are registered trademarks of Illumina, Inc.
AGILENT® is a registered trademark of Agilent Technologies
SPRISELECT® and AMPURE® are registered trademarks of Beckman Coulter, Inc.

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