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
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
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:
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
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 & Change Notifications
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]
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]
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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.