NEBNext Enzymatic Methyl-seq (EM-seq™) is a high-performance enzyme-based alternative to bisulfite conversion for the identification of 5mC and 5hmC. Unlike bisulfite conversion, this highly efficient method minimizes DNA damage, resulting in superior detection of methylated cytosines, with fewer sequencing reads.
The new NEBNext Enzymatic Methyl-seq v2 Conversion Module has a wider input range (as low as 100 pg) than the original EM-seq Conversion Module (NEB #E7125).
For use in Illumina® sequencing workflows, we recommend use of the NEBNext Enzymatic Methyl-seq v2 Kit (NEB #E8015) 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 NEBNext 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 also available.
View or download extensive EM-seq v2 performance data in our Data Supplement.
Product Information
View or download extensive EM-seq v2 performance data in our Data Supplement.
The NEBNext Enzymatic Methyl-seq v2 Conversion Module provides a high-performance enzyme-based alternative to bisulfite conversion for methylome analysis, with as little as 100 pg of input DNA.
EM-seq is a two-step enzymatic conversion process to detect modified cytosines. In the first step, TET2 and T4-BGT protect modified cytosines from downstream deamination. TET2 enzymatically oxidizes 5mC and 5hmC, and T4-BGT glucosylates 5hmC.
The second enzymatic step uses APOBEC to deaminate unmodified cytosines to uracils, but 5mC and 5hmC protected in the first step are not deaminated.
For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Conversion Module (NEB #E3365) is also available.
Figure 1: EM-seq™ conversion method
The EM-seq™ v2 workflow accommodates a wider input range than the original EM-seq workflow, with a 100-fold lower minimum input amount.
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
E8020S
-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
NEBNext® Carrier DNA
E3351AVIAL
-20
1 x 0.024 ml
Not Applicable
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
UDP-Glucose
E3353AVIAL
-20
1 x 0.024 ml
Not Applicable
DTT
E7139AAVIAL
-20
1 x 0.5 ml
100 mM
T4-BGT
E3354AVIAL
-20
1 x 0.024 ml
Not Applicable
T4-BGT Diluent
E8014AVIAL
-20
1 x 0.216 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
Deamination Reaction Buffer
E3356AVIAL
-20
1 x 0.096 ml
Not Applicable
Recombinant Albumin
E3357AVIAL
-20
1 x 0.024 ml
Not Applicable
E8020L
-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
NEBNext® Carrier DNA
E3351AAVIAL
-20
1 x 0.096 ml
Not Applicable
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
UDP-Glucose
E3353AAVIAL
-20
1 x 0.096 ml
Not Applicable
DTT
E7139AAVIAL
-20
1 x 0.5 ml
100 mM
T4-BGT
E3354AAVIAL
-20
1 x 0.096 ml
Not Applicable
T4-BGT Diluent
E8014AAVIAL
-20
1 x 0.864 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
Deamination Reaction Buffer
E3356AAVIAL
-20
1 x 0.384 ml
Not Applicable
Recombinant Albumin
E3357AAVIAL
-20
1 x 0.096 ml
Not Applicable
Properties & Usage
Materials Required but not Supplied
NEBNext UltraShear® (M7634) or Covaris® instrument and the required tubes or other fragmentation equipment
PCR strip tubes or 96-well plates
Cleanup beads: SPRIselect Reagent Kit (Beckman Coulter®, Inc. #B23317), AMPure® XP beads (Beckman Coulter, Inc. #A63881) or preferred bead manufacturer
Hi-Di™ Formamide (Thermo Fisher Scientific® #4401457), Formamide (Sigma #F9037-100 ml), or 0.05 N NaOH. Formamide is preferred. If using NaOH, please see FAQ associated with NEB #E8015.
80% Ethanol
10 mM Tris-HCl pH 7.5 or 8.0 or low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)
Nuclease-free Water
Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515)
Metal cooling block, such as Diversified Biotech® (#CHAM-1000)
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]
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 busdev@neb.com.
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 busdev@neb.com.