NEBNext® Enzymatic 5hmC-seq Kit
Product informationCode | Name | Size | Quantity | Price | |
---|---|---|---|---|---|
E3350S |
NEBNext Enzymatic 5hmC-seq Kit |
24 Assays | - | Unavailable in your region | |
E3350L |
NEBNext Enzymatic 5hmC-seq Kit |
96 Assays | - | Unavailable in your region |
NEBNext® Enzymatic 5hmC-seq Kit
Product Introduction
NEBNext® Enzymatic 5hmC-seq (E5hmC-seq™) is a new method for the specific detection of 5hmC at the single base level.
5hmC is detected using a two-step enzymatic conversion workflow that minimizes damage to DNA and allows discrimination of 5hmC from both cytosine and 5mC, after Illumina® sequencing. The kit has an input range of 0.1–200 ng and includes NEBNext Ultra™ II library prep reagents and the E5hmC-seq Adaptor.
Note that index primers are not included and must be purchased separately (NEBNext Primers for Epigenetics, NEB #E3392, NEB #E3404).
- High sensitivity of detection of 5hmC
- 0.1–200 ng input range
- Even GC coverage
- High-efficiency library preparation
- E5hmC-seq and EM-seq data can be combined
- Conversion module also available separately (NEB #E3365)
View or download extensive performance data below or in our Data Supplement.
Catalog # | Size | Concentration |
---|---|---|
E3350S | 24.0 reactions | |
E3350L | 96.0 reactions |
- Product Information
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Product Information
Description
View or download extensive performance data below or in our Data Supplement.
Typically, the modified cytosines 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are detected by sequencing of Illumina libraries generated using the NEBNext EM-seq enzyme-based workflow or bisulfite conversion. However, these methods cannot differentiate between 5mC and 5hmC.As with EM-seq, NEBNext Enzymatic 5hmC-seq (E5hmC-seq) uses an enzyme-based method to specifically detect 5hmC, without the DNA damage typical of bisulfite-based methods, and this sensitive method can be used with as little as 0.1 ng of input DNA.
In a two-step process, 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. This is followed by amplification using a NEBNext master mix formulation of Q5U® (a modified version of Q5® High-Fidelity DNA Polymerase), and sequencing on the Illumina platform.
Bioinformatic analysis tools used for EM-seq and for bisulfite sequencing can also be used for E5hmC-seq. E5hmC-seq data can be subtracted from EM-seq data, thereby allowing determination of the precise location of individual 5mC and 5hmC sites.
Note that index primers are not included and must be purchased separately (NEBNext Primers for Epigenetics, NEB #E3392, NEB #E3404).
- 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:
Properties & Usage
Materials Required but not Supplied
- Covaris® instrument and the required tubes or other fragmentation equipment
- NEBNext Primers for Epigenetics (Unique Dual Index Set 2B) NEB #E3392S (24 reactions) or Set 3 NEB #E3404S (96 reactions)
- PCR strip tubes or 96-well plates
- 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 #E3350 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
Related Products
Companion Products
Protocols, Manuals & Usage
Protocols
Manuals
Tools & Resources
Web Tools
FAQs & Troubleshooting
FAQs
- 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)?
- What are the recommended inputs for the NEBNext® Enzymatic 5hmC-seq Kit (NEB #E3350) and the NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)?
- What is the difference between the NEBNext® Enzymatic 5hmC-seq Kit (NEB #E3350) and the NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)?
- What buffers are recommended for shearing DNA when using the NEBNext Enzymatic 5hmC-seq Kit and the NEBNext 5hmC-seq Conversion Module?
- What is the concentration of the E5hmC-seq™ Adaptor and E5hmC-seq Index Primers?
- Why, at some stages of the E5hmC-seq™ protocol, do the NEBNext® Sample Purification Beads behave differently when cleaning up the sample?
- What is the expected size of an E5hmC-seq™ library?
- How should E5hmC-seq™ libraries be sequenced?
- How should E5hmC-seq™ sequencing data be analyzed?
- How deep should E5hmC-seq™ libraries be sequenced?
- Can the E5hmC-seq™ Adaptor be substituted with another adaptor?
- Can the E5hmC-seq™ Adaptor be used for EM-seq™?
- Are E5hmC-seq™ libraries directional or non-directional?
- Can other buffers be used in place of the supplied Elution Buffer?
- What levels of conversion are typical with the control DNAs supplied?
- Can enzymatically fragmented DNA be used in E5hmC-seq™?
- Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction step in the E5hmC-seq protocol?
- Do E5hmC-seq™ libraries detect 5mC?
- Are Sample Sheets available for use with the NEBNext® Primers for Epigenetics?
- Are dual-indexed libraries compatible with single end sequencing?
Citations & Technical Literature
Citations
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.Control DNA CpG unmethylated Lambda
NEBNext® Ultra II End Prep Reaction Buffer
NEBNext® Ultra II End Prep Enzyme Mix
NEBNext® Ultra™ II Ligation Master Mix
NEBNext® Ligation Enhancer
Elution Buffer
Stop Reagent
APOBEC
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 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
This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, 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.Other Products You May Be Interested In
The supporting documents available for this product can be downloaded below.