The NEBNext UltraExpress RNA Library Prep Kit is the latest generation of NEBNext RNA library prep, and it has a fast, streamlined workflow. With a 3-hour library prep protocol, the kit enables creation of high-quality RNA libraries in a single day, in conjunction with mRNA enrichment or rRNA depletion kits.
Fast workflow (3 hours)
Fewer steps and consumables
Fewer cleanups
Single protocol for all input amounts
High-quality libraries from a broad input range: 25 - 250 ng total RNA
Automation friendly
View or download extensive performance data below or in our Data Supplement.
This new kit meets the need for substantially faster, more streamlined RNA library prep workflows that deliver high-quality libraries from a variety of sample types in a single day from total RNA.
The kit allows the use of a single protocol for all RNA inputs (25 -250 ng total RNA) and incorporates master mixed reagents, reduced incubation times and fewer cleanup steps, reducing total time and consumables used.
3-hour library prep enables single day total RNA to library, in conjunction with mRNA enrichment or rRNA depletion kits
Fewer cleanups and pipetting steps reduce hands-on time
A robust single protocol for all input amounts increases ease of use
Flexibility is enabled with simple guidelines for customized protocols, if desired
Figure 2: The NEBNext UltraExpress™ RNA Library Prep Kit produces high library yields across a range of inputs
A. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent®), using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were prepared using the NEBNext UltraExpress™ RNA Library Prep Kit, Kapa mRNA HyperPrep® Kit or Illumina® Stranded mRNA Kit. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions (20X for 76–250ng, 100X for 25–75 ng) and PCR cycle numbers (listed in figure).
B. Ribosomal RNA (rRNA) was depleted from UHRR, and libraries were prepared using the UltraExpress RNA Library Prep Kit (preceded by the NEBNext rRNA Depletion Kit (Human/Mouse/Rat – NEB #E7400), Kapa HyperPrep Kit with RiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero® Plus. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 11 PCR cycles for all inputs, or with customized adaptor dilutions (20X for 76–250 ng, 100X for 25–75 ng) and PCR cycle numbers (listed in figure).
The total RNA input amount and number of PCR cycles are indicated. Library yields calculated from an average of three replicates are shown with error bars indicating the standard deviation between replicates.
A. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent®), using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were prepared using the NEBNext UltraExpress™ RNA Library Prep Kit, Kapa mRNA HyperPrep® Kit or Illumina® Stranded mRNA Kit. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent), and libraries were made using the UltraExpress RNA Library Prep Kit, Kapa mRNA HyperPrep Kit and Illumina Stranded mRNA Kit. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 10 PCR cycles for 250 ng input, 100X adaptor dilution with 12 PCR cycles (75 ng) and 14 PCR cycles (25 ng)).
B. Ribosomal RNA (rRNA) was depleted from UHRR, and libraries were prepared using the UltraExpress RNA Library Prep Kit (preceded by the NEBNext rRNA Depletion Kit (Human/Mouse/Rat – NEB #E7400), KAPA HyperPrep Kit with RiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero® Plus. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycles (20X adaptor dilution with 9 PCR cycles for 250 ng input, 100X adaptor dilution with 11 PCR cycles (75 ng) and 13 PCR cycles (25 ng)).
Libraries were sequenced on an Illumina NextSeq® 500 (2 x 75 bases). 10 million reads were sampled and mapped to the hg38 reference genome using RNA STAR v2.7.8a and 5´ to 3´ Transcript coverage was calculated from the top 1,000 transcripts using the CollectRnaSeqMetrics (Picard) tool v2.18.2.2.
Figure 4: The NEBNext UltraExpress™ RNA Library Prep Kit provides greater sensitivity of transcript detection
A. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent®), using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were prepared using the NEBNext UltraExpress™ RNA Library Prep Kit, Kapa mRNA HyperPrep® Kit or Illumina® Stranded mRNA Kit. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent), and libraries were made using the NEBNext UltraExpress RNA Library Prep Kit, Kapa mRNA HyperPrep Kit and Illumina Stranded mRNA Kit. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 10 PCR cycles for 250 ng input, 100X adaptor dilution with 12 PCR cycles (75 ng) and 14 PCR cycles (25 ng)).
B. Ribosomal RNA (rRNA) was depleted from UHRR, and libraries were prepared using the UltraExpress RNA Library Prep Kit (preceded by the NEBNext rRNA Depletion Kit (Human/Mouse/Rat – NEB #E7400), Kapa HyperPrep Kit with RiboErase, or Illumina Stranded Total RNA Library Prep Kit with Ribo-Zero® Plus. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 11 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 9 PCR cycles for 250 ng input, 100X adaptor dilution with 11 PCR cycles (75 ng) and 13 PCR cycles (25 ng)).
Libraries were sequenced on an Illumina NextSeq® 500 (2 x 75 bases). 10 million reads were sampled and average transcript abundance was assessed for transcripts detected with ≤ 10 reads and ≤ 500 reads, respectively. Error bars indicate standard deviation for 3 replicates. Salmon v1.5.1 was used for mapping and quantification of all gencode v38 transcripts and ERCCs.
Figure 5: The NEBNext UltraExpress™ RNA Library Prep Kit provides excellent transcript correlation between inputs and replicates
A. Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (UHRR) (Agilent®), using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries were prepared using the NEBNext UltraExpress RNA Library Prep with a single adaptor dilution (50X) and 12 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 10 PCR cycles for 250 ng input, 100X adaptor dilution with 12 PCR cycles (75 ng) and 14 PCR cycles (25 ng)).
B. Ribosomal RNA (rRNA) was depleted from UHRR using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat – NEB #E7400) and libraries. The NEBNext UltraExpress RNA Library Prep Kit was used with a single adaptor dilution (50X) and 11 PCR cycles for all inputs, or with customized adaptor dilutions and PCR cycle numbers (20X adaptor dilution with 9 PCR cycles for 250 ng input, 100X adaptor dilution with 11 PCR cycles (75 ng) and 13 PCR cycles (25 ng)).
Libraries were sequenced on an Illumina NextSeq® 500 (2 x 75 bases). The data was sampled from 10 million reads and libraries were correlated for transcript expression levels across inputs using Salmon v1.5.1 quantification of all gencode v38 transcripts and ERCCs. Each data point represents a transcript, with the log10 abundance in Number of Reads with 250 ng total RNA input on the y-axis compared to a replicate at 250 ng followed by 75 and 25 ng on the x-axis.
Figure 6: The NEBNext UltraExpress™ RNA Library Prep Kit provides excellent transcript correlation across sample types
RNA from human whole blood (HWB) was depleted of globin mRNA and rRNA (A) using the NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat – NEB #E7750) or rRNA was depleted from a pool of 20 different bacterial organisms (ATCC® #MSA-2002) (B) using the NEBNext rRNA Depletion Kit (Bacteria – NEB #E7850). Libraries were prepared using the NEBNext UltraExpress™ RNA Library Prep Kit, with customized adaptor dilution and PCR cycles per input (20X adaptor dilution with 9 PCR cycles for 250 ng input, 100X adaptor dilution with 11 PCR cycles (75 ng) and 13 PCR cycles (25 ng)). Libraries were sequenced on an Illumina® NextSeq® 500 (2 x 75 bases). The data was sampled from 10 million reads and libraries were correlated for transcript expression levels across inputs using Salmon v1.5.1 quantification of all gencode v38 transcripts (HWB) and a composite genome (Bacterial). Each data point represents a transcript, with the log10 abundance in number of Reads with 250 ng total RNA input on the y-axis compared to a replicate at 250 ng followed by 75 and 25 ng on the x-axis.
This product is related to the following categories:
NEBNext Multiplex Oligos options can be found at www.neb.com/oligos. Alternatively, customer supplied adaptor and primers can be used; please see information in link: https://www.neb.com/faqs/2019/03/08/can-i-use-this-nebnext-kit-with-adaptors-and-primers-from-other-vendors-than-neb
SPRIselect™ Reagent Kit (Beckman Coulter®, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
Magnetic Rack (NEB S1515S, Alpaqua® cat. #A001322, or equivalent)
80% Ethanol (freshly prepared)
Thermal cycler
DNase-, RNase-free PCR strip tubes, for example TempAssure® PCR flex-free 8-tube strips (USA Scientific® #1402-4708)
Bioanalyzer® or TapeStation® (Agilent® Technologies, Inc.) and associated reagents and consumables
For use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490):
1.5 ml Microcentrifuge tube and NEB #S1506 Magnet stand or equivalent (for washing beads only)
For use with NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400) and other NEBNext RNA depletion kits that do not include beads (NEB #E7750, E7850, E7865):
Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987)
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This product and its use are the subject of one or more issued and/or pending U.S. and foreign patent applications owned by Max Planck Gesellschaft. The purchase of this product from New England Biolabs, Inc., its affiliates, or its authorized resellers and distributors conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity) or for limited commercial purposes that are pre-approved by New England Biolabs, Inc. The purchase of this product does not convey a license under any claims in the foregoing patents or patent applications directed to producing the product. The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use this product for the following COMMERCIAL PURPOSES: (1) use of the products or its components in manufacturing; (2) use of the product or its components for therapeutic or prophylactic purposes in humans or animals.
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