Product Class: Kit

NEBNext® RNA Library Prep Kit for MGI®

Product Introduction

The NEBNext RNA Library Prep Kit for MGI is designed for use in MGI sequencing applications. This kit includes optimized components and protocols for the production of high-quality directional RNA libraries from a wide range of inputs (10 ng – 1 µg total RNA). The kit can be used with rRNA depletion or poly(A) enrichment protocols, including the Express protocol for the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490), as detailed in the product manual.

The NEBNext RNA Library Prep Kit for MGI is used in conjunction with NEBNext Multiplex Oligos for MGI (Dual Index Primer Pairs Set 1) (NEB #E9725) and libraries are circularized using the NEBNext Circularization Module for MGI (NEB #E9720) before sequencing.

  • Fast workflow
  • High library yields
  • Broad input range: 10 ng – 1 µg total RNA
  • Uniform transcript and GC coverage
  • Minimized adaptor-dimers

Request a sample of this product by visiting the NEBNext Sample Request Page and choosing “Sample not listed” at the bottom of the list; your local NEB Account Manager will reach out to confirm the details of your request.

 

Catalog # Size Concentration
E9710S 24 reactions
E9710L 96 reactions

Product Information

Description

The NEBNext RNA Library Prep Kit for MGI meets the need for reliable high-quality directional RNA library prep for MGI sequencing applications.

The kit incorporates trusted NEBNext master mixed reagents and protocols and allows the use of a wide range of inputs (10 ng – 1 µg total RNA). rRNA depletion or poly(A) enrichment protocols can be used upstream of the kit, and the Express protocol for the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) (see product manual) provides additional time savings.

Note that adaptors, primers and circularization reagents are provided separately, in the NEBNext Multiplex Oligos for MGI (Dual Index Primer Pairs Set 1) (NEB #E9725) and NEBNext Circularization Module for MGI (NEB #E9720).

  • Get more of what you need with high library yields
  • Generate high quality libraries with flexible input amounts from 10 ng to 1 µg total RNA
  • Increase the complexity and transcript coverage of your libraries
  • Optimize your time with streamlined workflows, minimized hands-on time, and automation compatibility
  • Rely on robust performance

 

FIGURE 1: NEBNext RNA Library Prep Kit for MGI workflow

E9710 Workflow

FIGURE 2: The NEBNext RNA Library Prep Kit for MGI produces high library yields across a range of inputs

E9710 MGI Yield


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 RNA Library Prep Kit for MGI using the recommendations for adaptor dilution and PCR cycles included in the kit protocol.

B. Ribosomal RNA (rRNA) was depleted from UHRR (NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400), and libraries were prepared using the NEBNext RNA Library Prep Kit for MGI, following the recommendations for adaptor dilution and PCR cycles included in the kit protocol. Library yields calculated from an average of three replicates are shown with error bars indicating the standard deviation between replicates.

FIGURE 3: The NEBNext RNA Library Prep Kit for MGI provides consistent transcript coverage

E7910 Transcript Coverage

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 RNA Library Prep Kit for MGI using the recommendations for adaptor dilution and PCR cycles included in the kit protocol.

B. Ribosomal RNA (rRNA) was depleted from UHRR (NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat), NEB #E7400), and libraries were prepared using the NEBNext RNA Library Prep Kit for MGI, following the recommendations for adaptor dilution and PCR cycles included in the kit protocol.  Libraries were sequenced on an MGI DNBSEQ-G400 (2 x 100 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. Even and consistent coverage was observed across all input amounts.


FIGURE 4: The NEBNext RNA Library Prep Kit for MGI provides high levels of detection for high- and low-abundance transcripts

E7910 High 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 RNA Library Prep Kit for MGI using the recommendations for adaptor dilution and PCR cycles included in the kit protocol.

B. Ribosomal RNA (rRNA) was depleted from UHRR (NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat), NEB #E7400), and libraries were prepared using the NEBNext RNA Library Prep Kit for MGI, following the recommendations for adaptor dilution and PCR cycles included in the kit protocol.  Libraries were sequenced on an MGI DNBSEQ-G400 (2 x 100 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. A high level of transcript detection was observed across all input amounts and the two workflows.


FIGURE 5: The NEBNext RNA library prep kit for MGI provides excellent transcript correlation between inputs and replicates

E7910 Transcript Correlation

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 RNA Library Prep Kit for MGI. The NEBNext RNA Library Prep Kit for MGI was used using the recommendations for adaptor dilution and PCR cycles included in the kit protocol.

B. Ribosomal RNA (rRNA) was depleted from UHRR (NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat), NEB #E7400) and libraries were prepared using the NEBNext RNA Library Prep Kit for MGI, following the recommendations for adaptor dilution and PCR cycles included in the kit protocol. Libraries were sequenced on an MGI DNBSEQ-G400 (2 x 100 bases). 10 million reads were sampled 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 Read Counts with 1 μg total RNA input on the y-axis compared to a replicate at 1 μg followed by 200 ng, 100 ng and 10 ng on the x-axis. The number of transcripts detected have very high correlation across the different inputs and replicates.

 
This product is related to the following categories:
Library Preparation for MGI®,
Next Generation Sequencing Library Preparation,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E9710S     -20    
      NEBNext® RNA Fragmentation Buffer E9618AVIAL -20 1 x 0.24 ml Not Applicable
      NEBNext First Strand Synthesis Enzyme Mix E7761AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext Strand Specificity Reagent E7766AVIAL -20 1 x 0.192 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® End Prep Reaction Buffer E9611AVIAL -20 1 x 0.168 ml Not Applicable
      NEBNext® End Prep Enzyme Mix E9612AVIAL -20 1 x 0.072 ml Not Applicable
      NEBNext® Ligation Master Mix E9613AVIAL -20 1 x 0.72 ml Not Applicable
      NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext USER® Enzyme E9619AVIAL -20 1 x 0.072 ml Not Applicable
      NEBNext® MSTC™ High Yield Master Mix E9615AVIAL -20 1 x 0.6 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
  • E9710L     -20    
      NEBNext® RNA Fragmentation Buffer E9618AAVIAL -20 1 x 0.96 ml Not Applicable
      NEBNext First Strand Synthesis Enzyme Mix E7761AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext Strand Specificity Reagent E7766AAVIAL -20 1 x 0.768 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® End Prep Reaction Buffer E9611AAVIAL -20 1 x 0.672 ml Not Applicable
      NEBNext® End Prep Enzyme Mix E9612AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Ligation Master Mix E9613AAVIAL -20 3 x 0.96 ml Not Applicable
      NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext USER® Enzyme E9619AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® MSTC™ High Yield Master Mix E9615AAVIAL -20 2 x 1.2 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

Properties & Usage

Materials Required but not Supplied

  • NEBNext Multiplex Oligos for MGI (Dual Index Primer Pairs Set 1) (#E9725)
  • NEBNext Circularization Module for MGI (#E9720)
  • 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 rRNA depletion kits that do not include beads (NEB #E7750, E7850, E78675):

  • Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987)

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for the  NEBNext® RNA Library Prep kit for MGI® (NEB #E9710)?

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

FAQs & Troubleshooting

FAQs

  1. What starting material must I use when preparing libraries with the NEBNext® RNA Library Prep Kit for MGI® (NEB #E9710)? 



  2. Which kit can I use to isolate poly(A) mRNA from total RNA?
  3. Which kit can I use to deplete ribosomal RNA (rRNA) from human, mouse or rat RNA?
  4. Which kit can I use to deplete ribosomal RNA (rRNA) from bacteria-derived RNA upstream of using the NEBNext® RNA Library Prep Kit for MGI® (NEB #E9710)?
  5. Is it possible to reduce the volume of beads added during the second strand synthesis cleanup step of the NEBNext® RNA Library Prep for MGI® (NEB # E9710) protocol?
  6. What do I do if I see a precipitate in the NEBNext® End Prep Reaction Buffer?
  7. The ligation reaction is very viscous. What will happen if it is not mixed properly?
  8. Do the NEBNext® Library Prep kits for MGI® include beads?
  9. Which kit can I use for circularization to make single-stranded circular DNA libraries for sequencing on MGI® platforms?
  10. How much library is required for the circularization reaction when using the NEBNext® Circularization Module for MGI® (NEB #E9635)?
  11. Do the NEBNext® library prep kits for MGI® include adaptors and primers?
  12. Can the NEBNext® RNA Library Prep Kit for MGI® (NEB #E9710) and NEBNext FS DNA Library Prep Kit for MGI (NEB #E9705) be used with adaptors and primers from suppliers other than NEB®?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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