Product Class: Kit

NEBNext® Ultra II RNA Library Prep Kit for Illumina®

Product Introduction

Note that Sample Purification beads are not included in this kit. If beads are required, we recommend ordering the NEBNext® Ultra II RNA Library Prep Kit for Illumina® (NEB #E7775).


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.



Bioz Badge Exists : False
Catalog # Size Concentration
E7770S 24 reactions 0 %
E7770L 96 reactions 0 %

Product Information

Description

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 5 ng or 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 with 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

Kit Components

The following reagents are supplied with this product:

Store at (°C) Concentration
NEBNext® Ligation Enhancer -20
NEBNext First Strand Synthesis Reaction Buffer -20
Random Primers -20
NEBNext Second Strand Synthesis Enzyme Mix -20
NEBNext Second Strand Synthesis Reaction Buffer -20 10 X
NEBNext Ultra II End Prep Enzyme Mix -20
NEBNext Ultra II End Prep Reaction Buffer -20
NEBNext® Ultra II Ligation Master Mix -20
NEBNext® Ultra II Q5® Master Mix -20 2 X
NEBNext First Strand Synthesis Enzyme Mix -20
NEBNext Adaptor Dilution Buffer -20 1 X
(0.1X) TE Buffer -20 0.1 X
Nuclease-free Water -20
Applications:
NEBNext® Ultra™ II RNA Library Prep,
RNA Library Preparation

Properties & Usage

Materials Required but not Supplied

  • NEBNext Singleplex (NEB #E7350) or NEBNext Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600) Oligos for Illumina or customer supplied oligos
  • Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)
  • SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
  • 80% Ethanol (freshly prepared)
  • Thermal Cycler
  • NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310)

Protocols, Manuals & Usage

Protocols

  1. Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770, E7775)
  2. Protocol for use with NEBNext® UltraTM II RNA Library Prep Kit for Illumina® E7770 and rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310)
  3. Protocol for use with FFPE RNA, NEBNext Ulta II RNA Library Prep Kit for Illumina E7770 and rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310)
  4. Protocols for use with Purified mRNA or rRNA Depleted RNA and NEBNext Ultra II RNA Library Prep Kit for Illumina
  5. Protocol for use with rRNA Depleted FFPE RNA and NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB # E7770, #E7775)
  6. Appendix A

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 the Monarch® Total RNA Miniprep Kit compatible with NEBNext® reagents for RNA Library Prep?
  2. What is the difference between the NEBNext Ultra II RNA Library Prep Kit (NEB #E7770) and the original NEBNext Ultra RNA Library Prep Kit (NEB #E7530)?
  3. What is the starting material I need to use when preparing libraries using the NEBNext Ultra II Directional RNA kit?
  4. Which kit can I use to isolate Poly (A) mRNA from total RNA?
  5. Which kit can I use to deplete ribosomal RNA (rRNA) from human, mouse or rat RNA?
  6. Does the kit include adaptor and primers?
  7. 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?
  8. Which NEBNext Oligos can be used with this library prep kit?
  9. Can I use this NEBNext kit with adaptors and primers from other vendors than NEB?

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]

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

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

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 [email protected].

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.