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

Description

Ultra II Sample
 
 

Ultra II Directional RNA Library Prep with Sample Purification Beads 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) enrichment, the kit enables the production of high quality libraries from 5 ng or 10 ng of Total RNA, respectively, up to 1µg.

This kit contains NEBNext Sample Purification Beads (SPRIselect® beads from Beckman Coulter) for size selection and enzyme reaction cleanup.

Strand-specific/directional methods for sequencing RNA provide information on the DNA strand from which the RNA strand was transcribed. This is useful for many reasons including: Identification of antisense transcripts, determination of the transcribed strand of noncoding RNA, and measurement of expression levels of coding or noncoding overlapping transcripts. Overall, the ability to determine the originating strand can substantially enhance the value of a RNA-seq experiment.

The NEBNext Ultra II Directional RNA Library Prep Kit derives its directionality from the “dUTP” method for strand-specificity, with proven superiority for this application.


Features

  • Get more of what you need, with the highest library yields
  • Generate high quality libraries even when you have only limited amounts of input RNA:
    • 10 ng – 1 µg Total RNA (polyA mRNA workflow)
    • 5 ng – 1 µg Total RNA (rRNA depletion workflow)
  • Minimize bias, with fewer PCR cycles required
  • Increase the complexity and transcript coverage of your libraries
  • Optimize your time with streamlined workflows, reduced hands-on time, and automation compatibility
  • Rely on robust performance, even with low quality RNA, including FFPE
  • Enjoy the flexibility and reliability of the gold standard SPRIselect size selection and clean-up beads, supplied in just the amounts you need
Also available without SPRIselect® beads for clean-up and size-selection steps.

Please note that adaptors, primers, rRNA depletion reagents and poly(A) mRNA isolation reagents are not included in the kit and are available separately.

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 Directional 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 made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Kapa Stranded mRNA-Seq Kit, Kapa mRNA HyperPrep Kit and Illumina TruSeq Stranded mRNA Kit. The input RNA amount and number of PCR cycles are indicated. Library yields from an average of three replicates are shown.

View additional data on library yields.



GC CONTENT DISTRIBUTION


Figure 2. NEBNext Ultra II Directional RNA libraries provide uniform GC content distribution, at a broad range of input amounts
CG Plot
Poly(A)-containing mRNA was isolated from Human Universal Reference RNA (Agilent #740000), and libraries were made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Illumina TruSeq Stranded mRNA Kit, Kapa Stranded mRNA-Seq Kit and Kapa mRNA HyperPrep Kit. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Reads were mapped to the hg19 reference genome. GC content distribution for each library was calculated using mapped reads. Ultra II Directional RNA libraries had uniform GC content distribution across a range of input amounts, whereas for other kits the GC content distribution changed with different input amounts, indicating the introduction of input-dependent sequence bias.

View additional data on library quality.



MAXIMIZING TRANSCRIPT COVERAGE


Figure 3. NEBNext Ultra II Directional 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 made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Illumina TruSeq Stranded mRNA Kit, Kapa Stranded mRNA-Seq Kit and Kapa mRNA HyperPrep Kit. 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 Directional RNA libraries as input RNA is decreased from 1 μg to 10 ng. The changes apparent in other kits result from loss of coverage at the 3´ end of some transcripts.

View additional data on transcript coverage.



SUPERIOR LIBRARY COMPLEXITY AT LOW INPUT AMOUNTS


Figure 4. Low input NEBNext Ultra II Directional RNA libraries retain superior complexity
Transcription
Poly(A)-containing mRNA was isolated from Human Universal Reference RNA (Agilent #740000), and libraries were made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Illumina TruSeq Stranded mRNA Kit, Kapa Stranded mRNA-Seq Kit and Kapa mRNA HyperPrep 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 GENCODE v25 transcripts. TPM = Transcripts Per Kilobase Million. R2 values for the linear fit are shown. Correlation analysis of the transcripts indicates superior transcript expression correlation between the different inputs for Ultra II Directional RNA libraries.
View additional data on library complexity.


SUPERIOR PERFORMANCE WITH FFPE RNA

Figure 5. NEBNext Ultra II Directional RNA with NEBNext rRNA Depletion results in the lowest remaining ribosomal RNA levels with FFPE samples
FFPE low
Ribosomal RNA was depleted from human adult normal liver tissue FFPE Total RNA (Biochain # R2234149. RIN 2.5) and libraries were made using NEBNext Ultra II Directional RNA Kit (plus the NEBNext rRNA Depletion Kit (Human/Mouse/Rat)), Kapa Stranded RNA-Seq Kit with RiboErase, Kapa HyperPrep Kit with RiboErase, and Illumina TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero™ Gold. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Read pairs were assessed to be rRNA if they contain 6 or more 32 base matches to 18S, 28S, 5S, 5.8S, 16S or 12S human rRNA sequences (mirabait 4.9). Percent rRNA remaining was calculated by dividing rRNA reads by the total number of reads passing instrument quality filtering. Average percent rRNA remaining is shown for three replicates. The NEBNext rRNA Depletion Ultra II Directional RNA workflow is the most efficient in removing rRNA from total FFPE RNA.


Figure 6. Uniformity of Coverage across the AP000769.1-201 transcript
FFPR ERRC
Ribosomal RNA was depleted from human adult normal liver tissue FFPE Total RNA (Biochain # R2234149. RIN 2.5), and libraries were made using NEBNext Ultra II Directional RNA Kit (plus the NEBNext rRNA Depletion Kit (Human/Mouse/Rat)), Illumina TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero™ Gold, Kapa Stranded RNA-Seq Kit with RiboErase and Kapa HyperPrep Kit with RiboErase. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Coverage across the length of this individual transcript (ENST00000625158.1; AP000769.1-201) was assessed by mapping reads directly to the GENCODE v25 transcripts and examining 100 bins along the transcript length. NEBNext Ultra II Directional RNA libraries provided coverage across the entire length of the transcript even as input was decreased from 100 ng to 10 ng.

View additional data on FFPE RNA samples.

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBNext First Strand Synthesis Enzyme Mix-20
NEBNext Strand Specificity Reagent-20
NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix-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 Ligation Enhancer-20
NEBNext USER Enzyme-20
NEBNext® Ultra II Q5® Master Mix-202X
NEBNext Adaptor Dilution Buffer-20
NEBNext® Sample Purification Beads25
NEBNext First Strand Synthesis Reaction Buffer-20
Random Primers-20
NEBNext Second Strand Synthesis Enzyme Mix-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)
  • 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 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?

Protocols

  1. Protocol for use with NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
  2. Protocol for use with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
  3. Protocol for use with FFPE RNA, NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
  4. Protocol for use with Purified mRNA or rRNA Depleted RNA and NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
  5. Protocol for use with rRNA Depleted FFPE RNA and NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
  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.