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

Even more from less, for RNA

Do you need increased sensitivity and specificity from your RNA-seq experiments? Do you have ever-decreasing amounts of input RNA? To address these challenges, our next generation of strand-specific RNA library prep kits have 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. The Ultra II Directional RNA kit uses the “dUTP method” (1, 2) for strand-specificity, and has a streamlined, automatable workflow.

Catalog # Concentration Size
E7765S Not Applicable 24 reactions
E7765L Not Applicable 96 reactions
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  • Product Information
    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.

    See what customers are saying about NEBNext Ultra II Directional RNA.

    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.

    This product is related to the following categories:
    RNA Library Prep for Illumina
    This product can be used in the following applications:
    NEBNext® Ultra™ II RNA Library Prep
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