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New England Biolabs

NEB NEBNextUltraII RNA Library Prep Kit for Illumina (Cat No. E7770)

NEB NEBNextUltraII RNA Library Prep Kit for Illumina (Cat No. E7770)

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For use in conjunction with this kit, the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) now has an Express protocol option for substantial time savings and a more streamlined workflow.

Note that Sample Purification beads are not included in this kit. If beads are required, we recommend ordering the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (NEB #E7775).


Do you prefer to use non-strand-specific library prep methods, but need increased sensitivity and specificity from your RNA-seq experiments, from ever-decreasing amounts of input RNA? To address these challenges, our next generation of non-directional RNA library prep kit has 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.



 

Catalog # Concentration Size
E7770S Not Applicable 24 reactions
E7770L Not Applicable 96 reactions

 

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.

See what customers are saying about NEBNext Ultra II RNA.


Features

Also available with optional 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 isolation or 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
Yield
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 yields


DUPLICATION RATES

Figure 2. NEBNext Ultra II RNA with NEBNext rRNA Depletion results in lower duplication rates .
Duplication
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 quality


MAXIMIZING TRANSCRIPT COVERAGE

Figure 3. NEBNext Ultra II 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 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 AMOUNTS

Figure 4. Low input NEBNext Ultra II RNA libraries retain complexity even at low input amounts
Complexity
Ribosomal 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

 

 

 

 

 

Product Name: NEB NEBNextUltraII RNA Library Prep Kit for Illumina (Cat No. E7770)

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