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 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.
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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
Compatible with NEBNext poly(A) mRNA Isolation, rRNA Depletion reagents and multiplexing adaptors and primers
Also available withoptional 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 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 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 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 yieldsDUPLICATION RATES Figure 2. NEBNext Ultra II RNA with NEBNext rRNA Depletion results in lower duplication rates .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 qualityMAXIMIZING TRANSCRIPT COVERAGE Figure 3. NEBNext Ultra II 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 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 AMOUNTSFigure 4. Low input NEBNext Ultra II RNA libraries retain complexity even at low input amountsRibosomal 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
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