NEBNext^® Enzymatic 5hmC-seq Kit

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
E3350S     Multi-temperature       NEBNext® Enzymatic 5hmC-seq Kit E3350-2 -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® Ultra II End Prep Reaction Buffer E7647AVIAL -20 1 x 0.168 ml Not Applicable   NEBNext® Ultra II End Prep Enzyme Mix E7646AVIAL -20 1 x 0.072 ml Not Applicable   NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml Not Applicable   NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml Not Applicable   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   NEBNext® E5hmC-seq™ Adaptor E3366AVIAL -20 1 x 0.06 ml Not Applicable   NEBNext® Q5U®  Master Mix E3369AVIAL -20 1 x 1.08 ml Not Applicable   NEBNext® Sample Purification Beads E3355S 25       NEBNext® Sample Purification Beads E3355AVIAL 25 1 x 5.2 ml Not Applicable
E3350L     Multi-temperature       NEBNext® Enzymatic 5hmC-seq Kit E3350-3 -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® Ultra II End Prep Reaction Buffer E7647AAVIAL -20 1 x 0.672 ml Not Applicable   NEBNext® Ultra II End Prep Enzyme Mix E7646AAVIAL -20 1 x 0.288 ml Not Applicable   NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml Not Applicable   NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml Not Applicable   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   NEBNext® E5hmC-seq™ Adaptor E3366AAVIAL -20 1 x 0.24 ml Not Applicable   NEBNext® Q5U®  Master Mix E3369AAVIAL -20 1 x 4.32 ml Not Applicable   NEBNext® Sample Purification Beads E3355L 25       NEBNext® Sample Purification Beads E3355AAVIAL 25 1 x 20.64 ml Not Applicable

Companion Products

• NEBNext^® Primers for Epigenetics (Unique Dual Index Set 2B)
• NEBNext^® Primers for Epigenetics (Unique Dual Index Set 3)
• NEBNext^® Enzymatic 5hmC-seq Conversion Module
• NEBNext^® Enzymatic Methyl-seq Kit

1. Where can I find protocols for use of the NEBNext^® Enzymatic 5hmC-seq Kit? (NEB #E3350)

• NEBNext Selector

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. What is the concentration of the E5hmC-seq™ Adaptor and E5hmC-seq Index Primers?
6. Why, at some stages of the E5hmC-seq™ protocol, do the NEBNext^® Sample Purification Beads behave differently when cleaning up the sample?
7. What is the expected size of an E5hmC-seq™ library?
8. How should E5hmC-seq™ libraries be sequenced?
9. How should E5hmC-seq™ sequencing data be analyzed?
10. How deep should E5hmC-seq™ libraries be sequenced?
11. Can the E5hmC-seq™ Adaptor be substituted with another adaptor?
12. Can the E5hmC-seq™ Adaptor be used for EM-seq™?
13. Are E5hmC-seq™ libraries directional or non-directional?
14. Can other buffers be used in place of the supplied Elution Buffer?
15. What levels of conversion are typical with the control DNAs supplied?
16. Can enzymatically fragmented DNA be used in E5hmC-seq™?
17. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction step in the E5hmC-seq protocol?
18. Do E5hmC-seq™ libraries detect 5mC?
19. Are Sample Sheets available for use with the NEBNext® Primers for Epigenetics?
20. Are dual-indexed libraries compatible with single end sequencing?

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.

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]

• E3350G_v1
• E3350L_v1
• E3350S_v1

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]

• E3350-1_v0_10230873
• E3350-2_v0_10230827
• E3350-3_v0_10222095

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

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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.