Next Ultra II Directional RNA Library Prep with Sample Purification Beads |NEB酶试剂 New England Biolabs

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产品信息

Next Ultra II Directional RNA Library Prep with Sample Purification Beads |
 
 

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 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)
    • 10 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
Next Ultra II Directional RNA Library Prep with Sample Purification Beads |
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
Next Ultra II Directional RNA Library Prep with Sample Purification Beads |
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 (2×76 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
Next Ultra II Directional RNA Library Prep with Sample Purification Beads |
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 (2×76 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
Next Ultra II Directional RNA Library Prep with Sample Purification Beads |
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 (2×76 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
Next Ultra II Directional RNA Library Prep with Sample Purification Beads |
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 (2×76 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
Next Ultra II Directional RNA Library Prep with Sample Purification Beads |
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 (2×76 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.

产品类别:
Automation for NEBNext® NGS Library Prep Products,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation Products

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E7765S     Multi-temperature    
        NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® E7760S -20    
        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 with dUTP Mix E7426AVIAL -20 1 x 0.192 ml Not Applicable
        NEBNext USER® Enzyme E7428AVIAL -20 1 x 0.072 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
        Nuclease-free Water E7764AVIAL -20 1 x 1.25 ml Not Applicable
        NEBNext Strand Specificity Reagent E7766AVIAL -20 1 x 0.192 ml Not Applicable
        NEBNext® Sample Purification Beads E7767S 25    
        NEBNext® Sample Purification Beads E7767SVIAL 25 1 x 6.65 ml Not Applicable
    • E7765L     Multi-temperature    
        NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® E7760L -20    
        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 with dUTP Mix E7426AAVIAL -20 1 x 0.768 ml Not Applicable
        NEBNext USER® Enzyme E7428AAVIAL -20 1 x 0.288 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
        Nuclease-free Water E7764AAVIAL -20 1 x 5.7 ml Not Applicable
        NEBNext Strand Specificity Reagent E7766AAVIAL -20 1 x 0.768 ml Not Applicable
        NEBNext® Sample Purification Beads E7767L 25    
        NEBNext® Sample Purification Beads E7767SVIAL 25 4 x 6.65 ml Not Applicable

  • 特性和用法

    需要但不提供的材料

    • 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)
    • 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 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)

操作说明、说明书 & 用法

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE7760_E7765

  • 应用实例

    • TechNote_NEBNext_Ultra_II_Directional_rRNA_Depletion
    • Obtain superior NGS library performance with lower input amounts using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®

FAQs & 问题解决指南

  • FAQs

    1. I have USER enzyme from my library prep kit blue cap and USER enzyme from my NEBNext Index Primer kit red cap Are they the same
    2. 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)?
    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. 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?

  • 问题解决指南

    • NEBNext® RNA Library Prep Kit Troubleshooting Guide (NEB #E7760, E7765, E7770, E7775, E7420, E7530)