NEBNext® Ultra II RNA Library Prep Kit for Illumina®

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 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.

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-2010X
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-202X
NEBNext First Strand Synthesis Enzyme Mix-20
NEBNext Adaptor Dilution Buffer-20
(0.1X) TE Buffer-200.1X
Nuclease-free Water-20

Properties and 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 (Alpaqua®, cat. #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)

FAQs

  1. 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)?
  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?

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® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra II RNA Library Prep Kit for Illumina® (E7770, E7775)
  3. Protocol for use with FFPE RNA, NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra II RNA Library Prep Kit for Illumina® (E7770, E7775)
  4. Protocol for use with Purified mRNA or rRNA Depleted RNA and NEBNext® Ultra II RNA Library Prep Kit for Illumina® (E7770, E7775)
  5. Protocol for use with rRNA Depleted FFPE RNA and NEBNext® Ultra II RNA Library Prep Kit for Illumina® (E7770, E7775)
  6. Appendix A for use with Ultra™ II RNA and Ultra II Directional RNA (E7760, E7765, E7770, E7775)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Troubleshooting Guides

Application Notes

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 Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.