The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
E7120S     Multi-temperature       NEBNext® Enzymatic Methyl-seq Kit E7120-2 -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® 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   Elution Buffer E7124AVIAL -20 1 x 2.1 ml Not Applicable   TET2 Reaction Buffer E7126AVIAL -20 1 x 0.3 ml Not Applicable   Oxidation Supplement E7128AVIAL -20 1 x 0.024 ml Not Applicable   Oxidation Enhancer E7129AVIAL -20 1 x 0.024 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   APOBEC Reaction Buffer E7134AVIAL -20 1 x 0.24 ml Not Applicable   BSA E7135AVIAL -20 1 x 0.024 ml Not Applicable   NEBNext® Q5U®  Master Mix E7136AVIAL -20 1 x 0.6 ml Not Applicable   DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM   TET2 Reaction Buffer Supplement E7127AVIAL -20 3 x 100 µl 100 X   NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) E7140S -20       EM-seq™ Index Primer 1 (S) E7141AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 2 (S) E7142AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 3 (S) E7143AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 4 (S) E7144AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 5 (S) E7145AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 6 (S) E7146AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 7 (S) E7147AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 8 (S) E7148AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 9 (S) E7149AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 10 (S) E7150AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 11 (S) E7151AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 12 (S) E7152AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 13 (S) E7153AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 14 (S) E7154AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 15 (S) E7155AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 16 (S) E7156AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 17 (S) E7157AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 18 (S) E7158AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 19 (S) E7159AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 20 (S) E7160AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 21 (S) E7161AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 22 (S) E7162AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 23 (S) E7163AVIAL -20 1 x 0.005 ml 10 µM   EM-seq™ Index Primer 24 (S) E7164AVIAL -20 1 x 0.005 ml 10 µM   NEBNext® EM-seq™ Adaptor E7165AVIAL -20 1 x 0.06 ml Not Applicable   NEBNext® Sample Purification Beads E7137S 25       NEBNext® Sample Purification Beads E7137AVIAL 25 1 x 8.6 ml Not Applicable
E7120L     Multi-temperature       NEBNext® Enzymatic Methyl-seq Kit E7120-3 -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® 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   Elution Buffer E7124AAVIAL -20 1 x 8.6 ml Not Applicable   TET2 Reaction Buffer E7126AAVIAL -20 1 x 1.2 ml Not Applicable   Oxidation Supplement E7128AAVIAL -20 1 x 0.096 ml Not Applicable   Oxidation Enhancer E7129AAVIAL -20 1 x 0.096 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   APOBEC Reaction Buffer E7134AAVIAL -20 1 x 0.96 ml Not Applicable   BSA E7135AAVIAL -20 1 x 0.096 ml Not Applicable   NEBNext® Q5U®  Master Mix E7136AAVIAL -20 2 x 1.2 ml Not Applicable   DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM   TET2 Reaction Buffer Supplement E7127AAVIAL -20 3 x 400 µl 100 X   NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) E7140L -20       NEBNext® 96 Unique Dual Index Primer Pairs Plate (L) E7166AVIAL -20 1 x 5 μl/well 10 µM   NEBNext® EM-seq™ Adaptor E7165AAVIAL -20 1 x 0.24 ml Not Applicable   NEBNext® Sample Purification Beads E7137L 25       NEBNext® Sample Purification Beads E7137AAVIAL 25 1 x 34.6 ml Not Applicable

1. It is possible for FE(II) Solution to have a yellow color. Testing has shown no difference in performance based on color.

1. Where can I find protocols for using the NEBNext^® Enzymatic Methyl-seq Kit? (NEB #E7120, E7125, E7140)

• E7140L_E7120L_EMSeqUDI_IlluminaLRM
• E7140S_E7120S_EMSeqUDI_IlluminaLRM
• Forward Strand Workflow Sample Sheet for E7140L: NovaSeq 6000 with v1.0 reagent kits, MiniSeq with Rapid reagent kits, MiSeq, HiSeq 2000/2500 (paired-end flow cell), HiSeq 3000/4000 (single-read flow cell)
• Forward Strand Workflow Sample Sheet for E7140S: NovaSeq 6000 with v1.0 reagent kits, MiniSeq with Rapid reagent kits, MiSeq, HiSeq 2000/2500 (paired-end flow cell), HiSeq 3000/4000 (single-read flow cell)
• Reverse Complement Workflow Sample Sheet for E7140L: iSeq 100, MiniSeq with Standard reagent kits, NextSeq Systems, NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/2500 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell)
• Reverse Complement Workflow Sample Sheet for E7140S: iSeq 100, MiniSeq with Standard reagent kits, NextSeq Systems, NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/2500 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell)

1. What types of sample can be processed using the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
2. What are the recommended inputs for the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
3. What is the difference between the NEBNext Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
4. What buffers are recommended for shearing DNA in NEBNext^® Enzymatic Methyl-seq (EM-seq^™) workflows?
5. What is the concentration of the EM-seq Adaptor and EM-seq Index Primers?
6. Can the “active” TET2 Buffer be stored longer than 4 months?
7. Can the freshly diluted Fe(II) Solution be stored long term?
8. Why, at some stages of the EM-seq protocol, do the NEBNext Sample Purification Beads behave differently when cleaning up the sample?
9. What is the expected size of an EM-seq library?
10. How should EM-seq libraries be sequenced?
11. How should EM-seq sequencing data be analyzed?
12. Can the EM-seq^™ Adaptor be substituted with another adaptor?
13. Are EM-seq^™ libraries directional or non-directional?
14. Can other buffers be used in place of the supplied EM-seq Elution Buffer?
15. How are EM-seq libraries prepared from cell-free DNA (cfDNA)?
16. Can EM-seq libraries be prepared from FFPE DNA?
17. What levels of conversion are typical with the control DNAs supplied in the EM-seq kit?
18. Are Sample Sheets available for use with the NEBNext® Multiplex Oligos for EM-seq?
19. Can enzymatically fragmented DNA be used as input material for EM-seq^™?
20. How should controls be diluted for applications involving shallow sequencing?
21. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction?
22. Are dual-indexed libraries compatible with single-end sequencing?
23. Is it normal if the Fe(II) solution from the EM-seq^™ product is yellow or a color change is observed?
24. What percentage of PhiX is needed for EM-seq^™ libraries?

• EM-seq Troubleshooting Guide

• Vaisvila R, et al. (2021) Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Res; PubMedID: 34140313, DOI: 10.1101/gr.266551.120

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]

• E7120G_v1
• E7120L_v1
• E7120S_v1

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

• Control DNA CpG methylated pUC19
• 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
• TET2 Reaction Buffer
• Oxidation Supplement
• Oxidation Enhancer
• TET2
• Fe (II) Solution
• Stop Reagent
• APOBEC
• APOBEC Reaction Buffer
• BSA
• NEBNext^® Q5U^®  Master Mix
• DTT
• TET2 Reaction Buffer Supplement
• EM-seq™ Index Primer 1 (S)
• EM-seq™ Index Primer 2 (S)
• EM-seq™ Index Primer 3 (S)
• EM-seq™ Index Primer 4 (S)
• EM-seq™ Index Primer 5 (S)
• EM-seq™ Index Primer 6 (S)
• EM-seq™ Index Primer 7 (S)
• EM-seq™ Index Primer 8 (S)
• EM-seq™ Index Primer 9 (S)
• EM-seq™ Index Primer 10 (S)
• EM-seq™ Index Primer 11 (S)
• EM-seq™ Index Primer 12 (S)
• EM-seq™ Index Primer 13 (S)
• EM-seq™ Index Primer 14 (S)
• EM-seq™ Index Primer 15 (S)
• EM-seq™ Index Primer 16 (S)
• EM-seq™ Index Primer 17 (S)
• EM-seq™ Index Primer 18 (S)
• EM-seq™ Index Primer 19 (S)
• EM-seq™ Index Primer 20 (S)
• EM-seq™ Index Primer 21 (S)
• EM-seq™ Index Primer 22 (S)
• EM-seq™ Index Primer 23 (S)
• EM-seq™ Index Primer 24 (S)
• NEBNext^® EM-seq™ Adaptor
• NEBNext^® Sample Purification Beads
• NEBNext^® 96 Unique Dual Index Primer Pairs Plate (L)

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.