Product Class: Kit

NEBNext® Enzymatic 5hmC-seq Conversion Module


Catalog #E3365

Product Introduction

NEBNext® Enzymatic 5hmC-seq (E5hmC-seq™) is a new method for the specific detection of 5hmC at the single base level.

The enzymatic conversion workflow minimizes damage to DNA and allows discrimination of 5hmC from both cytosine and 5mC

For E5hmC-seq in Illumina® sequencing workflows, we recommend use of the NEBNext Enzymatic 5hmC-seq Kit (NEB #E3350) instead of this module. The kit includes the E5hmC-seq Adaptor, as well as the high-efficiency NEBNext Ultra™ II library prep reagents, and these components are not included in the Conversion Module.

  • High-sensitivity specific detection of 5hmC
  • 0.1 – 200 ng input range
  • High efficiency conversion
  • Minimal DNA damage
  • E5hmC-seq kit including library prep reagents also available (NEB #E3350)

Product Information

Description

The NEBNext E5hmC-seq Conversion Module provides a high-performance enzyme-based method for detection of 5hmC at the single base level. In the E5hmC-seq two-step conversion workflow, T4-BGT glucosylates 5hmC, providing protection from deamination by APOBEC in the next step. T4-BGT does not protect 5mC or unmethylated cytosines, which are deaminated by APOBEC to uracils.

Note that for Illumina sequencing workflows, the NEBNext Enzymatic 5hmC-seq Kit, including library prep reagents is available (NEB #E3350)

Figure 1: E5hmC-seq conversion method

E3365 Conversion Module

To enable specific 5hmC detection, 5hmC is first glucosylated using T4-BGT. 5mC and unmodified cytosine are then deaminated by APOBEC to thymine and uracil, respectively, while the protected 5hmC is not converted.
This product is related to the following categories:
Methylome Analysis Products,
Next Generation Sequencing Library Preparation Products,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E3365S     -20    
      Control DNA CpG unmethylated Lambda E7123AVIAL -20 1 x 0.024 ml 2 ng/µl
      Control DNA 5hmC T4 E3349AVIAL -20 1 x 0.024 ml 1.5 ng/µl
      NEBNext® Carrier DNA E3351AVIAL -20 1 x 0.024 ml Not Applicable
      Elution Buffer E7124AVIAL -20 1 x 2.1 ml Not Applicable
      NEBNext® Glucosylation Reaction Buffer E3352AVIAL -20 1 x 0.12 ml Not Applicable
      UDP-Glucose E3353AVIAL -20 1 x 0.024 ml Not Applicable
      T4-BGT E3354AVIAL -20 1 x 0.024 ml Not Applicable
      Stop Reagent E7132AVIAL -20 1 x 0.024 ml Not Applicable
      Deamination Reaction Buffer E3356AVIAL -20 1 x 0.096 ml Not Applicable
      Recombinant Albumin E3357AVIAL -20 1 x 0.024 ml Not Applicable
      APOBEC E7133AVIAL -20 1 x 0.024 ml Not Applicable
  • E3365L     -20    
      Control DNA CpG unmethylated Lambda E7123AAVIAL -20 1 x 0.096 ml 2 ng/µl
      Control DNA 5hmC T4 E3349AAVIAL -20 1 x 0.096 ml 1.5 ng/µl
      NEBNext® Carrier DNA E3351AAVIAL -20 1 x 0.096 ml Not Applicable
      Elution Buffer E7124AAVIAL -20 1 x 8.6 ml Not Applicable
      NEBNext® Glucosylation Reaction Buffer E3352AAVIAL -20 1 x 0.48 ml Not Applicable
      UDP-Glucose E3353AAVIAL -20 1 x 0.096 ml Not Applicable
      T4-BGT E3354AAVIAL -20 1 x 0.096 ml Not Applicable
      Stop Reagent E7132AAVIAL -20 1 x 0.096 ml Not Applicable
      Deamination Reaction Buffer E3356AAVIAL -20 1 x 0.384 ml Not Applicable
      Recombinant Albumin E3357AAVIAL -20 1 x 0.096 ml Not Applicable
      APOBEC E7133AAVIAL -20 1 x 0.096 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • If DNA requires fragmentation: NEBNext UltraShear™ (NEB #M7634) or Covaris® instrument and the required tubes or other fragmentation equipment
  • PCR strip tubes or 96-well plates
  • Clean-up beads: SPRIselect™ Reagent Kit (Beckman Coulter®, Inc. #B23317), AMPure® XP beads (Beckman Coulter, Inc. #A63881) or preferred bead manufacturer.
  • Formamide (Sigma #F9037-100 ml), Hi-Di™ Formamide (Thermo Fisher Scientific® #4401457) or 0.1 N NaOH. Formamide is preferred. If using NaOH, please see FAQ on NEB #E3365 FAQ page.
  • 80% Ethanol
  • 1X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) or 10 mM Tris-HCl pH 7.5 or 8.0
  • Nuclease-free Water
  • Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515)
  • Metal cooling block, such as Diversified Biotech® (#CHAM-1000)
  • PCR machine
  • Agilent® Bioanalyzer®, TapeStation® or other fragment analyzer and associated consumables

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for use of the NEBNext® Enzymatic 5hmC-seq Conversion Module (NEB #E3365)?

Manuals

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

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What types of samples can be processed using the NEBNext® Enzymatic 5hmC-seq Kit (NEB #E3350) and the NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)?
  2. What are the recommended inputs for the NEBNext® Enzymatic 5hmC-seq Kit (NEB #E3350) and the NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)?
  3. What is the difference between the NEBNext® Enzymatic 5hmC-seq Kit (NEB #E3350) and the NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)?
  4. What buffers are recommended for shearing DNA when using the NEBNext Enzymatic 5hmC-seq Kit and the NEBNext 5hmC-seq Conversion Module?
  5. Why, at some stages of the E5hmC-seq™ Conversion Module protocol, do the NEBNext® Sample Purification Beads behave differently when cleaning up the sample?
  6. What levels of conversion are typical with the control DNAs supplied?
  7. Can enzymatically fragmented DNA be used in E5hmC-seq™?
  8. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction step in the E5hmC-seq protocol?

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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