The Ultra II Directional RNA Library Prep Kit for Illumina 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 1 µg.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 librarieseven 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 coverageof 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
Compatible with NEBNext poly(A) mRNA Isolation, rRNA Depletion reagents and multiplexing adaptors and primers.
Also available with optional 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 isolationor our technical note for rRNA depletion.
For use with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) (NEB #E7416), refer to the Protocols tab for UMI Adaptors-specific guidance.LIBRARY YIELDS
Figure 1. NEBNext Ultra II Directional RNA produces the highest yields, from a range of input amounts 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 DISTRIBUTIONFigure 2. NEBNext Ultra II Directional RNA libraries provide uniform GC content distribution, at a broad range of input amounts 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 transcriptsPoly(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 AMOUNTSFigure 4. Low input NEBNext Ultra II Directional RNA libraries retain superior complexityPoly(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 RNAFigure 5. NEBNext Ultra II Directional RNA with NEBNext rRNA Depletion results in the lowest remaining ribosomal RNA levels with FFPE samplesRibosomal 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 transcriptRibosomal 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:
Automation for NEBNext® NGS Library Prep,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation
This product can be used in the following applications:
Applications of USER® and Thermolabile USER II Enzymes
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