Product Class: Other

NEBNext® Enzymatic Methyl-seq Conversion Module

For improved performance, NEBNext Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020) is our recommendation.

Product Introduction

NEBNext® Enzymatic Methyl-seq (EM-seq™) is a new method for identification of 5-mC and 5-hmC.

While bisulfite sequencing has been the gold standard for methylome analysis, this conversion treatment damages DNA, resulting in fragmentation, loss and bias. The NEBNext Enzymatic Methyl-seq Conversion Module provides an enzyme-based alternative to bisulfite conversion.

For use in Illumina® sequencing workflows, we recommend use of the NEBNext Enzymatic Methyl-seq Kit (NEB #E7120) instead of this module. The full kit includes the EM-seq Adaptor, which has been optimized for high performance in the EM-seq workflow, as well as the high-efficiency Ultra II library prep reagents, and these components are not included in the Conversion Module.

For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Conversion Module (NEB #E3365) is now also available.

Catalog # Size Concentration
E7125S 24 reactions
E7125L 96 reactions

Product Information

Description

Download Technical Note

The NEBNext Enzymatic Methyl-seq Conversion Module provides a high-performance enzyme-based alternative to bisulfite conversion for methylome analysis.

In the EM-seq workflow, TET2 oxidizes 5-mC and 5-hmC, providing protection from deamination by APOBEC in the next step. In contrast, unmodified cytosines are deaminated to uracils.  

Features:

  • High efficiency conversion
  • Minimal DNA damage
  • Minimized GC bias
  • 5-mC and 5-hmC detection
  • Complete kit including library prep reagents also available (NEB #E7120)

For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Conversion Module (NEB #E3365) is now also available.



Figure 1: NEBNext Enzymatic Methyl-seq and Sodium Bisulfite Conversion Methods





This product is related to the following categories:
Automation for NEBNext® NGS Library Prep,
Enzymatic Conversion for DNA Methylation Analysis,
DNA Methylation Analysis,
Epigenetics,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7125S     -20    
      Control DNA CpG methylated pUC19 E7122AVIAL -20 1 x 0.024 ml 0.1 ng/µl
      Control DNA CpG unmethylated Lambda E7123AVIAL -20 1 x 0.024 ml 2 ng/µl
      Elution Buffer E7124AVIAL -20 1 x 2.1 ml Not Applicable
      TET2 Reaction Buffer E7126AVIAL -20 1 x 0.3 ml Not Applicable
      TET2 Reaction Buffer Supplement (x 1) E8013AVIAL -20 3 x 100 µl Not Applicable
      Oxidation Supplement E7128AVIAL -20 1 x 0.024 ml Not Applicable
      Oxidation Enhancer E7129AVIAL -20 1 x 0.024 ml Not Applicable
      TET2 E7130AVIAL -20 1 x 0.096 ml Not Applicable
      Fe (II) Solution E7131AVIAL -20 1 x 0.024 ml Not Applicable
      Stop Reagent E7132AVIAL -20 1 x 0.024 ml Not Applicable
      APOBEC E7133AVIAL -20 1 x 0.024 ml Not Applicable
      APOBEC Reaction Buffer E7134AVIAL -20 1 x 0.24 ml Not Applicable
      BSA E7135AVIAL -20 1 x 0.024 ml Not Applicable
      100mM DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM
  • E7125L     -20    
      Control DNA CpG methylated pUC19 E7122AAVIAL -20 1 x 0.096 ml 0.1 ng/µl
      Control DNA CpG unmethylated Lambda E7123AAVIAL -20 1 x 0.096 ml 2 ng/µl
      Elution Buffer E7124AAVIAL -20 1 x 8.6 ml Not Applicable
      TET2 Reaction Buffer E7126AAVIAL -20 1 x 1.2 ml Not Applicable
      TET2 Reaction Buffer Supplement (x 1) E8013AAVIAL -20 3 x 400 µl Not Applicable
      Oxidation Supplement E7128AAVIAL -20 1 x 0.096 ml Not Applicable
      Oxidation Enhancer E7129AAVIAL -20 1 x 0.096 ml Not Applicable
      TET2 E7130AAVIAL -20 1 x 0.384 ml Not Applicable
      Fe (II) Solution E7131AAVIAL -20 1 x 0.096 ml Not Applicable
      Stop Reagent E7132AAVIAL -20 1 x 0.096 ml Not Applicable
      APOBEC E7133AAVIAL -20 1 x 0.096 ml Not Applicable
      APOBEC Reaction Buffer E7134AAVIAL -20 1 x 0.96 ml Not Applicable
      BSA E7135AAVIAL -20 1 x 0.096 ml Not Applicable
      100mM DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM

Properties & Usage

Materials Required but not Supplied

  • PCR strip tubes
  • Formamide (Sigma #F9037-100 ml) or 0.1 N NaOH
  • 80% Ethanol
  • 0.1X TE, pH 8.0
  • Nuclease-free Water
  • Clean-up Beads: NEBNext Sample Purification Beads (supplied with Enzymatic Methyl-seq Kit, NEB #E7120), SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317), AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or preferred bead manufacturer.
  • Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515)
  • PCR machine
  • Bioanalyzer®, TapeStation® and associated consumables or other fragment analyzer

Product Notes

  1. It is possible for FE(II) Solution to have a yellow color. Testing has shown no difference in performance based on color.

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using the NEBNext® Enzymatic Methyl-seq Kit? (NEB #E7120, E7125, E7140)

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 types of sample can be processed using the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
  2. What are the recommended inputs for the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
  3. What is the difference between the NEBNext Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
  4. What buffers are recommended for shearing DNA in NEB’s enzymatic methyl-seq (EM-seq™) workflows?
  5. Can the “active” TET2 Buffer be stored longer than 4 months?
  6. Can the freshly diluted Fe(II) Solution be stored long term?
  7. Why, at some stages of the EM-seq protocol, do the NEBNext Sample Purification Beads behave differently when cleaning up the sample?
  8. Can enzymatically fragmented DNA be used as input material for EM-seq™?
  9. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction?
  10. Is it normal if the Fe(II) solution from the EM-seq product is yellow or a color change is observed?
  11. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction?
  12. Is it normal if the Fe(II) solution from the EM-seq™ product is yellow or a color change is observed?

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.

Licenses

In the United States and Europe, this product cannot be used in, or incorporated into, any blood-based, sequencing-based screening diagnostic assay (including product or service-based offerings) for the early detection of colorectal, gastric, lung, liver, ovarian, breast, prostate, esophageal, thyroid, uterine, pancreatic, bladder or kidney cancer indications.

This product and/or its manufacture and/or its use either alone or in combination with other product(s) are protected by one or more of the following patents and related pending patents: U.S. Pat. Nos. 9,121,061, 9,896,726, 10,227,646, 10,260,088, 10,619,200, 11,001,876, and 11,124,825; European Pat. Nos. EP2825645 and EP3368688; JP6224689; and CN108699598.

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. No. 8,470,573 and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows. For more information, please email [email protected].

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