INSTRUCTION MANUAL
NEBNext® Enzymatic Methyl-seq Kit
NEB #E7120S/L

24/96 reactions
Version 7.0_4/23

Table of Contents
Overview ..............................................................................................................................................................................................2
Section 1
Protocol for use with Standard Size Libraries (370–420 bp) ...............................................................................................................4
Section 2
Protocol for use with Large Size Libraries (470–520 bp)..................................................................................................................11
Section 3
Index Guidelines ................................................................................................................................................................................18
Kit Components .................................................................................................................................................................................23
Checklist (Standard Size Libraries) ...................................................................................................................................................25
Checklist (Large Size Libraries) ........................................................................................................................................................29
Revision History ................................................................................................................................................................................33

The Library Kit Includes
The volumes provided are sufficient for preparation of up to 24 reactions (NEB #E7120S) and 96 reactions (NEB #E7120L).
The NEBNext Sample Purification Beads should be stored at room temperature and all other reagents should be stored at –20°C.
Colored bullets represent the color of the cap of the tube containing the reagent.
(lilac) Control DNA CpG methylated pUC19(lilac) Control DNA Unmethylated Lambda(green) NEBNext Ultra™ II End Prep Reaction Buffer(green) NEBNext Ultra II End Prep Enzyme Mix(red) NEBNext Ultra II Ligation Master Mix(red) NEBNext Ligation Enhancer(red) NEBNext EM-seq™ Adaptor(white) Elution Buffer(yellow) TET2 Reaction Buffer(yellow) TET2 Reaction Buffer Supplement(yellow) Oxidation Supplement(yellow) DTT(yellow) Oxidation Enhancer(yellow) TET2
(yellow) Fe(II) Solution (Please Note: This buffer may be clear or yellow in color.)(yellow) Stop Reagent(orange) APOBEC(orange) APOBEC Reaction Buffer(orange) BSA(blue) NEBNext Q5U™ Master Mix
NEBNext Multiplex Oligos for Enzymatic Methyl-seq (24 Unique Dual Index Primer Pairs) or (96 Unique Dual Index Primer Pairs)
NEBNext Sample Purification Beads

1

Required Materials Not Included•

Covaris® S2 instrument or other fragmentation equipment•

PCR strip tubes•

Recommended: Formamide (Sigma #F9037-100 ml) or optional 0.1 N NaOH. Formamide is preferred.
If using NaOH, please see FAQ on NEB #E7120 FAQ page.

•

80% Ethanol•

1XTE (10 mM Tris, 1 mM EDTA, pH 8.0), 10 mM Tris pH 8.0, Low TE (10 mM Tris, 0.1 mM EDTA, pH 8.0) or 0.1XTE (1mM
Tris, 0.1 mM EDTA, pH 8.0).

•

Nuclease-free Water•

Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515S)•

PCR machine•

Bioanalyzer®, TapeStation® and associated consumables or other fragment analyzer

Overview
Figure 1. NEBNext Enzymatic Methyl-seq Kit Workflow.

The Enzymatic Methyl-seq kit (EM-seq) for Illumina contains all the components needed to make libraries that are enzymaticallymodified to detect 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC).
Figure 1 is an overview of the EM-seq workflow. Firstly, a library is made by ligating EM-seq adaptor to sheared end repaired/dA-tailed
genomic DNA. This is followed by two sets of enzymatic conversion steps to differentiate unmethylated cytosines from 5mC/5hmC.
Finally, libraries are PCR amplified before sequencing.

2

Figure 2. Overview of Sodium Bisulfite Conversion and EM-seq.

Figure 2 shows a comparison of the sodium bisulfite and EM-seq methods. Sodium bisulfite treatment of DNA results in the deaminationof cytosines into uracils, however the modified forms of cytosine (5mC and 5hmC) are not deaminated. Therefore, the preference ofbisulfite to chemically deaminate cytosines enables the methylation status of cytosines to be determined. When bisulfite treated DNA is
PCR amplified, uracils are replaced by thymines and the 5mC/5hmC are replaced by cytosines. Once sequenced, unmethylated cytosines
are represented by thymines and 5mC and 5hmC are represented by cytosines. By comparing sequences to non-converted genomes theappropriate methylation status can be assessed.
Enzymatic Methyl-seq is a two step enzymatic conversion process to detect modified cytosines. The first step uses TET2 and an oxidation
enhancer to protect modified cytosines from downstream deamination. TET2 enzymatically oxidizes 5mC and 5hmC through a cascade
reaction into 5-carboxycytosine [5-methylcytosine (5mC) 5-hydroxymethylcytosine (5hmC)  5-formylcytosine (5fC)  5carboxycytosine (5caC)]. This protects 5mC and 5hmC from deamination. 5hmC can also be protected from deamination by glucosylation
to form 5ghmc using the oxidation enhancer. The second enzymatic step uses APOBEC to deaminate C but does not convert 5caC and5ghmC. The resulting converted sequence can be analyzed like bisulfite-treated DNA. Typical aligners used to analyze data include but are
not limited to Bismark and BWAMeth.
The workflow described in the NEBNext Enzymatic Methyl-seq Kit is user-friendly and enables methylation detection from inputs ranging
between 10 ng–200 ng. EM-seq converted DNA is more intact than bisulfite-converted DNA, resulting in libraries with longer sequencing
reads, reduced GC bias and more even genome coverage.
Please note that the bead volumes provided are sufficient for building standard size libraries described in Section 1, Protocol for use withStandard Size Libraries (370–420 bp). If following the Section 2 Protocol, for use with Large Size Libraries (470–520 bp), users need tosupply additional beads due to the increased volumes needed for cleanups. We recommend using SPRIselect® Reagent Kit (Beckman
Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881). If you are combining leftover beads from E7120 withthe above products, we recommend combing with SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317).
Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated
together by construction of indexed libraries and sequenced on an Illumina sequencing platform.
For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM & Custom Solutions
department at NEB. Please contact custom@neb.com for further information.

3

Section 1
Protocol for use with Standard Libraries (370–420 bp)
Symbols

This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
This caution sign signifies a step in the protocol that has two paths leading to the same end point.
Colored bullets indicate the cap color of the reagent to be added.
Starting Material: 10 ng–200 ng DNA
1.1. DNA Preparation
1.1.1.DNA and Control DNA
DNA and Control DNA
Combine genomic DNA (10–200 ng) with control DNAs specified below. Genomic DNA can be in any of the following buffers:
10 mM Tris pH 8.0, 1X TE (10mM Tris pH 8.0, 1mM EDTA), or low TE (10 mM Tris pH 8.0, 0.1 mM EDTA).
COMPONENTVOLUME

gDNA

48 µl

(lilac) Control DNA Unmethylated Lambda (see Table 1.1)

1 µl(lilac) Control DNA CpG methylated pUC19 (see Table 1.1)

1 µl

Total Volume

50 µl

The following table is a guide for the amount of (lilac) Control DNA CpG methylated pUC19 and (lilac) Control DNA
Unmethylated Lambda DNA to be added to samples prior to EM-seq library construction. Expected read numbers along withread length should be considered to ensure that enough controls are included for the users individual sequencing goals.
Table 1.1 Dilutions of control DNAs for a range of genomic DNA inputs. These are suitable for shallow/pre-sequencing(approx. 2 – 4 million paired reads) on a MiSeq prior to deep sequencing (approx. 100-150 million paired reads) on
NovaSeq, HiSeq or NextSeq.
DILUTION OF (LILAC) LAMBDA CONTROL AND (LILAC) pUC19 CONTROLDNA
Input AmountPre-sequencing

Deep Sequencing

2–4 Million Paired Reads

100–150 Million Paired Reads

10 ng

1:20

1:100

200 ng

No Dilution

1:50

Regardless of sequencing depth, a minimum of 5,000 paired end reads with a read length of 76 bases, for unmethylated Lambda
DNA, and 500 paired end reads with a read length of 76 bases, for CpG methylated pUC19, are needed to give enough coverage
for accurate conversion estimates.
Different applications may require different sequencing depths and therefore different strategies should be employed when
deciding how much control DNA should be added. For example, some libraries may only need 2 million paired end reads
whereas other may require 50 million paired end reads or even 300 million paired end reads.
Additional considerations, regarding the amount of controls added, should be taken into account. For example, pre-sequencing
libraries to a depth of 2-4 million paired end reads using the recommended dilution for the controls (Table 1.1), followed bydeeper sequencing of these same libraries to a higher depth of 100 – 150 million paired reads per library would result in excess
reads associated with the controls. This strategy is recommended users who choose to check library conversion prior to deeper
sequencing. Users who dilute controls based on pre-sequencing guidelines will have excess control reads, thus ensuring a higher
confidence in library conversion.

4

1.1.2.Shearing DNA
The combined 50 µl genomic DNA and control DNAs are fragmented to an average fragment size of ~300 bp (370–420 bp final
Illumina library). Fragmentation can be done using a preferred fragmentation device such as a Covaris instrument. Enzymatic
fragmentation is not recommended as this may result in the removal of methylation marks.
Transfer the 50 µl of sheared DNA to a new PCR tube for End Prep.
Note: DNA does not need to be cleaned up or size selected before End Prep
1.2.End Prep of Sheared DNA
1.2.1.

On ice, mix the following components in a sterile nuclease-free PCR tube:
COMPONENT
Sheared DNA (Step 1.1.2.)

VOLUME
50 µl(green) NEBNext Ultra II End Prep Reaction Buffer

7 µl(green) NEBNext Ultra II End Prep Enzyme Mix

3 µl

Total Volume

60 µl

Note: NEBNext Ultra II End Prep Reaction Buffer and Enzyme Mix can be pre-mixed ahead of time as a master mix.
1.2.2.

Set a 100 µl or 200 µl pipette to 50 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly.
Perform a quick spin to collect all liquid from the sides of the tube.
Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with the performance.

1.2.3.

Place in a thermal cycler with the heated lid set to ≥ 75°C or on, and run the following program:
30 minutes at 20°C
30 minutes at 65°C
Hold at 4°C

1.3.Ligation of EM-seq Adaptor
1.3.1.

On ice, add the following components directly to the End Prep reaction mixture and mix well:
COMPONENTVOLUMEEnd Prep reaction mixture (Step 1.2.3.)

60 µl(red) NEBNext EM-seq Adaptor

2.5 µl(red) NEBNext Ligation Enhancer

1 µl(red) NEBNext Ultra II Ligation Master Mix

30 µl

Total Volume

93.5 µlNote: Ligation Enhancer and Ligation Master Mix can be mixed ahead of time and is stable for at least 8 hours at 4°C. Do
not premix the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step. Premix
adaptor and sample and then add the other ligation reagents.
1.3.2.

Set a 100 µl or 200 µl pipette to 80 µl and then pipette the entire volume up and down 10 times to mix thoroughly. Perform aquick spin to collect all liquid from the sides of the tube.
Caution: The Ligation Master Mix is viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as
incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interferewith performance.

1.3.3.

Place in a thermal cycler, and run the following program with the heated lid off:
15 minutes at 20°C
Hold at 4°C

Safe Stopping Point: Samples can be stored overnight at -20°C.
1.4.Clean-Up of Adaptor Ligated DNA
1.4.1.

Vortex Sample Purification Beads to resuspend.

1.4.2.

Add 110 µl (~1.1X) of resuspended NEBNext Sample Purification Beads to each sample. Mix well by pipetting up and down atleast 10 times. Be careful to expel all of the liquid out of the tip during the last mix.

5

1.4.3.

Incubate samples on bench top for at least 5 minutes at room temperature.

1.4.4.

Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.

1.4.5.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads
that contain DNA targets (Caution: do not discard the beads).

1.4.6.

Add 200 μl of 80% freshly prepared ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

1.4.7.

Repeat the ethanol wash once for a total of two washes. Be sure to remove all visible liquid after the second wash using a p10
pipette tip.

1.4.8.

Air dry the beads for up to 2 minutes while the tubes are on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads
are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brownand start to crack they are too dry.

1.4.9.

Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 29 µl of Elution Buffer (white).

1.4.10.

Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at room temperature. If necessary, quickly spin thesample to collect the liquid from the sides of the tube before placing back on the magnetic stand.

1.4.11.

Place the tube on the magnetic stand. After 3 minutes (or whenever the solution is clear), transfer 28 µl of the supernatant to a
new PCR tube.

Safe Stopping Point: Samples can be stored overnight at -20°C.
1.5.Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
1.5.1.

Prepare TET2 Buffer. Use option A if you have E7120S/E7120G (24 reactions/G size) and option B if you have E7120L (96reactions).
Note: The (yellow) TET2 Reaction Buffer Supplement is a powder. Centrifuge before use to ensure it is at the bottom of
the tube.

1.5.1A.

Add 100 µl of (yellow) TET2 Reaction Buffer to one tube of (yellow) TET2 Reaction Buffer Supplement and mix well.
Write date on tube.

1.5.1B.

Add 400 µl of (yellow) TET2 Reaction Buffer to one tube of (yellow) TET2 Reaction Buffer Supplement and mix well.
Write date on tube.
Note: The reconstituted buffer should be stored at -20°C and discarded after 4 months.

1.5.2.

On ice, add the following components directly to the EM-seq adaptor ligated DNA
COMPONENTVOLUME

EM-seq adaptor ligated DNA (from Step 1.4.11)

28 µl(yellow) TET2 Reaction Buffer (TET2 Reaction Buffer
plus reconstituted TET2 Reaction Buffer Supplement)

10 µl(yellow) Oxidation Supplement

1 µl(yellow) DTT

1 µl(yellow) Oxidation Enhancer

1 µl(yellow) TET2

4 µl

Total Volume

45 µl

Mix thoroughly by vortexing, centrifuge briefly. For multiple reactions, a master mix of the above reaction components can beprepared before addition to the sample DNA. 5mC/5hmC oxidation is initiated by the addition of the Fe(II) solution to thereaction in the next step.
1.5.3.

Dilute the 500 mM(yellow) Fe(II) Solution by adding 1 µl to 1,249 µl of water.

Note: The 500 mM (yellow) Fe(II) solution color can vary between colorless to yellow, this is normal. Use the diluted
solution immediately, do not store it. Discard after use.

6

Combine diluted(yellow) Fe(II) Solution and Reaction Mixture DNA with Oxidation Enzymes as described below:

COMPONENTVOLUME

Reaction Mixture (Step 1.5.2)

45 µl

Diluted (yellow) Fe(II) Solution (Step 1.5.3)

5 µl

Total Volume

50 µl

Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
1.5.4.

Place in a thermal cycler, and run the following program with the heated lid set to  45C or on:
1 hour at 37°C
Hold at 4°C

1.5.5.

Transfer the samples to ice and add 1 µl of(yellow) Stop Reagent.

COMPONENTVOLUME

Oxidized DNA (Step 1.5.4.)

50 µl(yellow) Stop Reagent

1 µl

Total Volume

51 µl

Mix thoroughly by vortexing or by pipetting up and down at least 10 times and centrifuge briefly.
1.5.6.

Place in a thermal cycler, and run the following program with the heated lid set to  45C or on:
30 minutes at 37°C
Hold at 4°C

Safe Stopping Point: Samples can be stored overnight at either 4°C in the thermal cycler or at -20°C in the freezer.
1.6. Clean-Up of TET2 Oxidized DNA
1.6.1.

Vortex Sample Purification Beads to resuspend.

1.6.2.

Add 90 µl (1.8X) of resuspended NEBNext Sample Purification Beads to each sample. Mix well by pipetting up and down atleast 10 times. Be careful to expel all of the liquid out of the tip during the last mix.

1.6.3.

Incubate samples on bench top for at least 5 minutes at room temperature.

1.6.4.

Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.

1.6.5.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads
that contain DNA targets (Caution: do not discard the beads).

1.6.6.

Add 200 μl of 80% freshly prepared ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

1.6.7.

Repeat the wash once for a total of two washes. Be sure to remove all visible liquid after the second wash using a p10 pipette tip.

1.6.8.

Air dry the beads for up to 2 minutes while the tubes are on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads
are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brownand start to crack they are too dry.

1.6.9.

Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 17 µl of

1.6.10.

Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at room temperature. If necessary, quickly spin thesample to collect the liquid from the sides of the tube before placing back on the magnetic stand.

(white) Elution Buffer.

1.6.11.

Place the tube on the magnetic stand. After 3 minutes (or whenever the solution is clear), transfer 16 µl of the supernatant to a
new PCR tube.

Safe Stopping Point: Samples can be stored overnight at -20°C.

71.7. Denaturation of DNA

The DNA can be denatured using either Formamide or 0.1 N Sodium Hydroxide.
Use option A for denaturing using Formamide and option B for denaturing using 0.1 N Sodium hydroxide.
1.7A:

Formamide (Recommended)

1.7A.1.

Pre-heat thermal cycler to 85°C with the heated lid set to ≥ 105°C or on.

1.7A.2.

Add 4 µl Formamide to the 16 µl of oxidized DNA (Step 1.6.11). Vortex to mix or by pipetting up and down at least 10 times,
centrifuge briefly.

1.7A.3.

Incubate at 85°C for 10 minutes in the pre-heated thermal cycler.

1.7A.4.

Immediately place on ice and allow the sample to fully cool (~2 min) before proceeding to Section 1.8.

1.7B:

Sodium Hydroxide (Optional, See FAQ about preparing NaOH)

1.7B.1.

Prepare freshly diluted 0.1 N NaOH.

1.7B.2.

Pre-heat thermal cycler to 50°C with the heated lid set to ≥ 105°C or on.

1.7B.3.

Add 4 µl 0.1 N NaOH to the 16 µl of oxidized DNA (Step 1.6.11). Vortex to mix or by pipetting up and down at least 10 times,
centrifuge briefly.

1.7B.4.

Incubate at 50°C for 10 minutes in the pre-heated thermal cycler.

1.7B.5.

Immediately place on ice and allow the sample to fully cool (~2 min) before proceeding to Section 1.8.

1.8. Deamination of Cytosines
1.8.1.

On ice, add the following components to the denatured DNA:COMPONENTVOLUME

Denatured DNA (Step 1.7A.4. or 1.7B.5.)

20 µl

Nuclease-free water

68 µl(orange) APOBEC Reaction Buffer

10 µl(orange) BSA

1 µl(orange) APOBEC

1 µl

Total Volume

100 µlFor multiple reactions, a master mix of the reaction components can be prepared before addition to the denatured DNA.
1.8.2.

Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.

1.8.3.

Place in a thermal cycler, and run the following program with the heated lid set to  45C or on:
3 hours at 37°C
Hold at 4°C

Safe Stopping Point: Samples can be stored overnight at either 4°C in the thermal cycler or at -20°C in the freezer.
1.9. Clean-Up of Deaminated DNA
Caution: The Sample Purification Beads behave differently during the APOBEC clean-up. After the bead washes, do not overdry
the beads as they become very difficult to resuspend.
1.9.1.

Vortex Sample Purification Beads to resuspend.

1.9.2.

Add 100 µl (1.0X) of resuspended NEBNext Sample Purification Beads to each sample. Mix well by pipetting up and down atleast 10 times. Be careful to expel all of the liquid out of the tip during the last mix.

1.9.3.

Incubate samples on bench top for at least 5 minutes at room temperature.

1.9.4.

Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.

1.9.5.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads
that contain DNA targets (Caution: do not discard the beads).

8

1.9.6.

Add 200 μl of 80% freshly prepared ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

1.9.7.

Repeat the wash once for a total of two washes. Be sure to remove all visible liquid after the second wash using a p10 pipette tip.

1.9.8.

Air dry the beads for up to 90 seconds while the tubes are on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads
are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brownand start to crack they are too dry.

1.9.9.

Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 21 µl of (white) Elution Buffer.

1.9.10.

Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at room temperature. If necessary, quickly spin thesample to collect the liquid from the sides of the tube before placing back on the magnetic stand.

1.9.11.

Place the tube on the magnetic stand. After 3 minutes (or whenever the solution is clear), transfer 20 µl of the supernatant to a
new PCR tube.

Safe Stopping Point: Samples can be stored overnight at -20°C.
1.10.PCR Amplification
1.10.1.

On ice, add the following components to the deaminated DNA:COMPONENTVOLUME

Deaminated DNA (Step 1.9.11.)

20 µl

EM-seq Index Primer*, **

5 µl
25 µl

(blue) NEBNext Q5U Master Mix
Total Volume

50 µl*Refer to Section 3 for barcode pooling guidelines.
** EM-seq primers are supplied in tubes in NEB #E7120S or as a 96 Unique Dual Index Primers Pairs Plate in NEB #E7120L.
1.10.2.

Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.

1.10.3.

Place the tube in a thermal cycler with the heated lid set to 105°C and perform PCR amplification using the following cycling
conditions:
CYCLE STEP

TEMP

TIME

CYCLES

Initial Denaturation

98°C

30 seconds

1

Denaturation

98°C

10 seconds

Annealing

62°C

30 seconds

Extension

65°C

60 seconds

Final Extension

65°C

Hold

4°C

5 minutes∞*Cycle Recommendations:• 10 ng DNA input:• 50 ng DNA input:• 200 ng DNA input:

4–8*
1

8 cycles5-6 cycles
4 cyclesSafe Stopping Point: Samples can be stored overnight at either 4°C in the thermal cycler or at -20°C in the freezer.
1.11.Clean-Up of Amplified Libraries
1.11.1.

Vortex Sample Purification Beads to resuspend.

1.11.2.

Add 45 µl (0.9X) of resuspended NEBNext Sample Purification Beads to each sample. Mix well by pipetting up and down atleast 10 times. Be careful to expel all of the liquid out of the tip during the last mix.

1.11.3.

Incubate samples on bench top for at least 5 minutes at room temperature.

1.11.4.

Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.

9

1.11.5.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads
that contain DNA targets (Caution: do not discard the beads).

1.11.6.

Add 200 μl of 80% freshly prepared ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

1.11.7.

Repeat the wash once for a total of two washes. Be sure to remove all visible liquid after the second wash using a p10 pipette tip.

1.11.8.

Air dry the beads for up to 2 minutes while the tubes are on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads
are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brownand start to crack they are too dry.

1.11.9.

Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 21 µl of (white) Elution Buffer. For
long terms storage use, 21 µl of 1XTE (10 mM Tris, 1 mM EDTA, pH 8.0), Low TE (10 mM Tris, 0.1 mM EDTA, pH 8.0) or
0.1XTE (1mM Tris, 0.1 mM EDTA, pH 8.0).

1.11.10. Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at room temperature. If necessary, quickly spin thesample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
1.11.11. Place the tube on the magnetic stand. After 3 minutes (or whenever the solution is clear), transfer 20 µl of the supernatant to a
new PCR tube.

Safe Stopping Point: Samples can be stored overnight at -20°C.
1.12.Library Quantification
1.12.1.

Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries. A typical EM-seq librarywould have the following TapeStation trace.

50 ng of NA12878 genomic DNA

Sequence using the preferred Illumina platform. 2 x 76 base reads or 2 x
100 base reads for standard sized libraries.

10Section 2
Protocol for use with Large Size Libraries (470–520 bp)
Symbols

This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
This caution sign signifies a step in the protocol that has two paths leading to the same end point.
Colored bullets indicate the cap color of the reagent to be added.
Starting Material: 10 ng–200 ng DNA.
2.1.DNA Preparation
2.1.1.
DNA and Control DNA
Combine genomic DNA (10–200 ng) with control DNAs specified below. Genomic DNA can be in any of the following buffers:
10 mM Tris pH 8.0, 1X TE (10mM Tris pH 8.0, 1mM EDTA), or low TE (10 mM Tris pH 8.0, 0.1 mM EDTA).
COMPONENTVOLUME

gDNA

48 µl

(lilac) Control DNA Unmethylated Lambda (see Table 2.1)

1 µl

(lilac) Control DNA CpG methylated pUC19 (see Table 2.1)

1 µl

Total Volume

50 µl

The following table is a guide for the amount of (lilac) Control DNA CpG methylated pUC19 and (lilac) Control DNA
Unmethylated Lambda DNA to be added to samples prior to EM-seq library construction. Expected read numbers along withread length should be considered to ensure that enough controls are included for the users individual sequencing goals.
Table 2.1 Dilutions of control DNAs for a range of genomic DNA inputs. These are suitable for shallow/pre-sequencing(approx. 2 - 4 million paired reads) on a MiSeq prior to deep sequencing (approx. 100-150 million paired reads) on
NovaSeq, HiSeq or NextSeq.
DILUTION OF (LILAC) LAMBDA CONTROLAND (LILAC) pUC19 CONTROLDNA
Input AmountPre-sequencing

Deep Sequencing

2-4 Million Paired Reads

100-150 Million Paired Reads

10 ng

1:20

1:100

200 ng

No Dilution

1:50

Regardless of sequencing depth, a minimum of 5,000 paired end reads with a read length of 76 bases, for unmethylated Lambda
DNA, and 500 paired end reads with a read length of 76 bases, for CpG methylated pUC19, are needed to give enough coverage
for accurate conversion estimates.
Different applications may require different sequencing depths and therefore different strategies should be employed when
deciding how much control DNA should be added. For example, some libraries may only need 2 million paired end reads
whereas other may require 50 million paired end reads or even 300 million paired end reads.
Additional considerations, regarding the amount of controls added, should be taken into account. For example, pre-sequencing
libraries to a depth of 2-4 million paired end reads using the recommended dilution for the controls (Table 2.1), followed bydeeper sequencing of these same libraries to a higher depth of 100 – 150 million paired reads per library would result in excess
reads associated with the controls. This strategy is recommended users who choose to check library conversion prior to deeper
sequencing. Users who dilute controls based on pre-sequencing guidelines will have excess control reads, thus ensuring a higher
confidence in library conversion.

11

2.1.2.Shearing DNA
The combined 50 µl genomic DNA and control DNAs are fragmented to an average fragment size of ~500 bp (470–520 bp final
Illumina library). Fragmentation can be done using a preferred fragmentation device such as a Covaris instrument. Enzymatic
fragmentation is not recommended as this may result in the removal of methylation marks.
Transfer the 50 µl of sheared DNA to a new PCR tube for End Prep.
Note: DNA does not need to be cleaned up or size selected before End Prep
2.2. End Prep of Sheared DNA
2.2.1.

On ice, mix the following components in a sterile nuclease-free PCR tube:
COMPONENTVOLUME

Fragmented DNA

50 µl(green) NEBNext Ultra II End Prep Reaction Buffer

7 µl
3 µl

(green) NEBNext Ultra II End Prep Enzyme Mix
Total Volume

60 µl

Note: NEBNext Ultra II End Prep Reaction Buffer and Enzyme Mix can be pre-mixed ahead of time as a master mix.
2.2.2

2.2.3.

Set a 100 µl or 200 µl pipette to 50 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly.
Perform a quick spin to collect all liquid from the sides of the tube. Note: It is important to mix well. The presence of a small
amount of bubbles will not interfere with the performance.
Place in a thermal cycler with the heated lid set to ≥ 75°C or on, and run the following program:
30 minutes at 20°C
30 minutes at 65°C
Hold at 4°C

2.3.

Ligation of EM-seq Adaptor

2.3.1.

On ice, add the following components directly to the End Prep reaction mixture and mix well:
COMPONENTVOLUMEEnd Prep reaction mixture (Step 2.2.3.)

60 µl(red) NEBNext EM-seq Adaptor

2.5 µl(red) NEBNext Ligation Enhancer

1 µl(red) NEBNext Ultra II Ligation Master Mix

30 µl

Total Volume

93.5 µlNote: Ligation Enhancer and Ligase Master Mix can be mixed ahead of time and is stable for at least 8 hours at 4°C. We
do not recommend adding adaptor to a premix in the adaptor ligation step. Premix adaptor and sample and then add theother ligation reagents.
2.3.2.

Set a 100 µl or 200 µl pipette to 80 µl and then pipette the entire volume up and down 10 times to mix thoroughly. Perform aquick spin to collect all liquid from the sides of the tube.
Caution: The Ligase Master Mix is viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as
incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interferewith performance.

2.3.3.

Place in a thermal cycler, and run the following program with the heated lid off:
15 minutes at 20°C
Hold at 4°C

Safe Stopping Point: Samples can be stored overnight at -20°C.
2.4. Clean-Up of Adaptor Ligated DNA
2.4.1.

Vortex Sample Purification Beads to resuspend.

2.4.2.

Add 110 µl (~1.1X) of resuspended NEBNext Sample Purification Beads to each sample. Mix well by pipetting up and down atleast 10 times. Be careful to expel all of the liquid out of the tip during the last mix.

2.4.3.

Incubate samples on bench top for at least 5 minutes at room temperature.

12

2.4.4.

Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.

2.4.5.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads
that contain DNA targets (Caution: do not discard the beads).

2.4.6.

Add 200 μl of 80% freshly prepared ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

2.4.7.

Repeat the ethanol wash once for a total of two washes. Be sure to remove all visible liquid after the second wash using a p10
pipette tip.

2.4.8.

Air dry the beads for up to 2 minutes while the tubes are on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads
are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brownand start to crack they are too dry.

2.4.9.

Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 29 µl of Elution Buffer (white).

2.4.10.

Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at room temperature. If necessary, quickly spin thesample to collect the liquid from the sides of the tube before placing back on the magnetic stand.

2.4.11.

Place the tube on the magnetic stand. After 3 minutes (or whenever the solution is clear), transfer 28 µl of the supernatant to a
new PCR tube.

Safe Stopping Point: Samples can be stored overnight at -20°C.
2.5. Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
2.5.1.

Prepare TET2 Buffer. Use option A if you are using E7120S/E7120G (24 reactions/G size) and option B if you are using E7120L(96 reactions).
Note: Thethe tube.

(yellow) TET2 Reaction Buffer Supplement is a powder. Centrifuge before use to ensure it is at the bottom of

2.5.1A.

Add 100 µl of (yellow) TET2 Reaction Buffer to one tube of
mix well. Write date on tube.

(yellow) TET2 Reaction Buffer Supplement and2.5.1B.

Add 400 µl of (yellow) TET2 Reaction Buffer to one tube of
mix well. Write date on tube.

(yellow) TET2 Reaction Buffer Supplement andNote: The reconstituted buffer should be stored at -20°C and discarded after 4 months.
2.5.2.

On ice, add the following components directly to the EM-seq adaptor ligated DNA:COMPONENTVOLUME

Em-Seq Adaptor ligated DNA (Step 2.4.11.)28 µl(yellow) TET2 Reaction Buffer (TET2 Reaction Buffer
plus reconstituted TET2 Reaction Buffer Supplement)

10 µl(yellow) Oxidation Supplement

1 µl(yellow) DTT

1 µl(yellow) Oxidation Enhancer

1 µl(yellow) TET2

4 µl

Total Volume

45 µl

Mix thoroughly by vortexing, centrifuge briefly. For multiple reactions, a master mix of the above reaction components can beprepared before addition to the sample DNA. 5mC/5hmC oxidation is initiated by the addition of the Fe(II) solution to thereaction in the next step.
2.5.3.

Dilute the 500 mM(yellow) Fe(II) Solution by adding 1 µl to 1,249 µl of water.

Note: The 500 mM (yellow) Fe(II) solution color can vary between colorless to yellow, this is normal. Use the diluted
solution immediately, do not store it. Discard after use.
Combine diluted(yellow) Fe(II) Solution and EM-seq DNA with Oxidation Enzymes (from Step 2.5.2).

13

COMPONENTVOLUME

Reaction mixture (Step 2.5.2)

45 µl

Diluted5 µl(yellow) Fe(II) Solution (Step 2.5.3)

Total Volume

50 µl

Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
2.5.4.

Place in a thermal cycler, and run the following program with the heated lid set to  45C or on:
1 hour at 37°C
Hold at 4°C

2.5.5.

Transfer the samples to ice and add(yellow) Stop Reagent:

COMPONENTVOLUME

Oxidized DNA (Step 2.5.4.)

50 µl(yellow) Stop Reagent

1 µl

Total Volume

51 µl

Mix thoroughly by vortexing or by pipetting up and down at least 10 times and centrifuge briefly.
2.5.6.

Place in a thermal cycler, and run the following program with the heated lid set to  45C or on:
30 minutes at 37°C
Hold at 4°C

Safe Stopping Point: Samples can be stored overnight at either 4°C in the thermal cycler or at -20°C in the freezer.
2.6. Clean-Up of TET2 Oxidized DNA
2.6.1.

Vortex Sample Purification Beads to resuspend.

2.6.2.

Add 90 µl (1.8X) of resuspended NEBNext Sample Purification Beads to each sample. Mix well by pipetting up and down atleast 10 times. Be careful to expel all of the liquid out of the tip during the last mix.

2.6.3.

Incubate samples on bench top for at least 5 minutes at room temperature.

2.6.4.

Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.

2.6.5.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads
that contain DNA targets (Caution: do not discard the beads).

2.6.6.

Add 200 μl of 80% freshly prepared ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

2.6.7.

Repeat the wash once for a total of two washes. Be sure to remove all visible liquid after the second wash using a p10 pipette tip.

2.6.8.

Air dry the beads for up to 2 minutes while the tubes are on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads
are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brownand start to crack they are too dry.

2.6.9.

Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 17 µl of(white) Elution Buffer.

2.6.10.

Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at room temperature. If necessary, quickly spin thesample to collect the liquid from the sides of the tube before placing back on the magnetic stand.

2.6.11.

Place the tube on the magnetic stand. After 3 minutes (or whenever the solution is clear), transfer 16 µl of the supernatant to a
new PCR tube.

Safe Stopping Point: Samples can be stored overnight at -20°C.

14

2.7. Denaturation of DNA

The DNA can be denatured using either Formamide or 0.1 N Sodium Hydroxide.
Use option A for denaturing using Formamide and option B for denaturing using 0.1 N Sodium hydroxide.
2.7A:

Formamide (Recommended)

2.7A.1.

Pre-heat thermal cycler to 85°C with the heated lid set to ≥ 105°C or on.

2.7A.2.

Add 4 µl Formamide to the 16 µl of oxidized DNA (Step 2.6.11). Vortex to mix or by pipetting up and down at least 10 times,
centrifuge briefly.

2.7A.3.

Incubate at 85°C for 10 minutes in the pre-heated thermal cycler.

2.7A.4.

Immediately place on ice and allow the sample to fully cool (~2 min) before proceeding to Section 2.8.

2.7B:

Sodium Hydroxide (Optional, see FAQ about preparing NaOH)

2.7B.1.

Prepare freshly diluted 0.1 N NaOH.

2.7B.2.

Pre-heat thermal cycler to 50°C with the heated lid set to ≥ 105°C or on.

2.7B.3.

Add 4 µl 0.1 N NaOH to the 16 µl of oxidized DNA (Step 2.6.11). Vortex to mix or by pipetting up and down at least 10 times,
centrifuge briefly.

2.7B.4.

Incubate at 50°C for 10 minutes in the pre-heated thermal cycler.

2.7B.5.

Immediately place on ice and allow the sample to fully cool (~2 min) before proceeding to Section 2.8.

2.8.

Deamination of Cytosines

2.8.1.

On ice, add the following components to the denatured DNA:COMPONENTVOLUME

Denatured DNA (Step 2.7A.4. or 2.7B.5.)

20 µl

Nuclease-free water

68 µl(orange) APOBEC Reaction Buffer

10 µl(orange) BSA

1 µl(orange) APOBEC

1 µl

Total Volume

100 µlFor multiple reactions, a master mix of the reaction components can be prepared before addition to the denatured DNA.
2.8.2.

Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.

2.8.3.

Place in a thermal cycler, and run the following program with the heated lid set to  45C or on:
3 hours at 37°C
Hold at 4°C

Safe Stopping Point: Samples can be stored overnight at either 4°C in the thermal cycler or at -20°C in the freezer.
2.9. Clean-Up of Deaminated DNA
Caution: The Sample Purification Beads behave differently during the APOBEC clean up. After the bead washes, do not overdry
the beads as they become very difficult to resuspend.
2.9.1.

Vortex Sample Purification Beads to resuspend.

2.9.2.

Add 100 µl (1.0X) of resuspended NEBNext Sample Purification Beads to each sample. Mix well by pipetting up and down atleast 10 times. Be careful to expel all of the liquid out of the tip during the last mix.

2.9.3.

Incubate samples on bench top for at least 5 minutes at room temperature.

2.9.4.

Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.

2.9.5.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads
that contain DNA targets (Caution: do not discard the beads).

2.9.6.

Add 200 μl of 80% freshly prepared ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

15

2.9.7.

Repeat the wash once for a total of two washes. Be sure to remove all visible liquid after the second wash using a p10 pipette tip.

2.9.8.

Air dry the beads for up to 90 seconds while the tubes are on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads
are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brownand start to crack they are too dry.

2.9.9.

Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 21 µl of(white) Elution Buffer.

2.9.10.

Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at room temperature. If necessary, quickly spin thesample to collect the liquid from the sides of the tube before placing back on the magnetic stand.

2.9.11.

Place the tube on the magnetic stand. After 3 minutes (or whenever the solution is clear), transfer 20 µl of the supernatant to a
new PCR tube.

Safe Stopping Point: Samples can be stored overnight at -20°C.
2.10.PCR Amplification
2.10.1. On ice, add the following components to the deaminated DNA:COMPONENTVOLUME

Deaminated DNA (Step 2.9.11.)

20 µl

EM-seq Index Primer *,**

5 µl
25 µl

(blue) NEBNext Q5U Master Mix
Total Volume

50 µl*Refer to Section 3 for barcode pooling guidelines.
** EM-seq primers are supplied in tubes in NEB #E7120S or as a 96 Unique Dual Index Primers Pairs Plate in NEB #E7120L.
2.10.2.

Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.

2.10.3.

Place the tube in a thermal cycler with the heated lid set to 105°C and perform PCR amplification using the following cycling
conditions:
CYCLE STEP

TEMP

TIME

CYCLES

Initial Denaturation

98°C

30 seconds

1

Denaturation

98°C

10 seconds

Annealing

62°C

30 seconds

Extension

65°C

60 seconds

Final Extension

65°C

Hold

4°C

5 minutes
∞

4–8*

1

* Cycle Recommendations:• 10 ng DNA input:• 50 ng DNA input:• 200 ng DNA input:

8 cycles5-6 cycles
4 cyclesSafe Stopping Point: Samples can be stored overnight at either 4°C in the thermal cycler or at -20°C in the freezer.
2.11.Clean-Up of Amplified Libraries
2.11.1.

Vortex Sample Purification Beads to resuspend.

2.11.2.

Add 90 µl of water to each sample. Mix well by pipetting up and down at least 10 times.

2.11.3.

Add 91 µl of resuspended NEBNext Sample Purification Beads to each sample. Mix well by pipetting up and down at least 10
times. Be careful to expel all of the liquid out of the tip during the last mix.

2.11.4.

Incubate samples on bench top for at least 5 minutes at room temperature.

2.11.5.

Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.

16

2.11.6.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads
that contain DNA targets (Caution: do not discard the beads).

2.11.7.

Add 200 μl of 80% freshly prepared ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

2.11.8.

Repeat the ethanol wash once for a total of two washes. Be sure to remove all visible liquid after the second wash using a p10
pipette tip.

2.11.9.

Air dry the beads for up to 2 minutes while the tubes are on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads
are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brownand start to crack they are too dry.

2.11.10. Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 21 µl of (white) Elution Buffer. For
long terms storage use, 21 µl of 1XTE (10 mM Tris, 1 mM EDTA, pH 8.0), Low TE (10 mM Tris, 0.1 mM EDTA, pH 8.0) or
0.1XTE (1mM Tris, 0.1 mM EDTA, pH 8.0).
2.11.11. Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at room temperature. If necessary, quickly spin thesample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
2.11.12. Place the tube on the magnetic stand. After 3 minutes (or whenever the solution is clear), transfer 20 µl of the supernatant to a
new PCR tube.

Safe Stopping Point: Samples can be stored overnight at -20°C.
2.12.Library Quantification
2.12.1.

Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries. A typical EM-seq librarywould have the following TapeStation trace.

50 ng of NA12878 genomic DNA.

Sequence using the preferred Illumina platform. 2 x 100 base reads or 2 x 150 base reads
for large insert libraries.

17

Section 3
Index Pooling Guidelines
For more detailed indexing information please refer to the manual for NEBNext Multiplex Oligos for Enzymatic Methyl-seq (Unique DualIndex Primer Pairs), NEB #E7140.
For a link to download a sample sheet with the index sequences for use with the Illumina Experiment Manager (IEM) please go
to our FAQ's tab on www.neb.com/E7140 – NEBNext Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer
Pairs), NEB #E7140.
Adaptor trimming sequences:
The NEBNext libraries for Illumina resemble TruSeq libraries and can be trimmed similar to TruSeq:
Adaptor Read 1 AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
Adaptor Read 2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

24 Reaction Kit (NEB #E7120S)
For multiplexing < 24 samples use Table 3.1 for some valid index combinations.
Table 3.1.
PLEX

TUBE NUMBER
1 and 2*2

3 and 45 and 6
7 and 83

Any 2 plex plus any other index* Use only one primer tube per library18

* Forward Strand Workflow for the following instruments: NovaSeq 6000 with v1.0 reagents kits, MiniSeq with rapid reagent kits, MiSeq®,
HiSeq® 2000/2500 (pair-end flow cell), HiSeq 3000/4000 (single-read flow cell).
* Reverse Complement Workflow for the following instruments: iSeq 100, MiniSeq with standard reagent kits, NextSeq Systems,
NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/5000 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell).
Note: Please see Illumina Document “Indexed Sequencing Overview” document number 15057455
The index primer sequences, for different Illumina sequencer input sheets are indicated in Table 3.2.
Table 3.2 Index Sequences (Color coded based on HiSeq/MiSeq guidelines)
TUBE
NUMBEREXPECTED i7 INDEX READ

EXPECTED i5 INDEX READ
Forward Strand Workflow*

Reverse Complement Workflow*

1

CACTGTAG

AAGCGACTAGTCGCTT

2GTGCACGATGATAGGCGCCTATCA

3

AAGCGACTACGAATCC

GGATTCGT

4TGATAGGC

GTCTGAGTACTCAGAC5ACGAATCCATTACCCATGGGTAAT

6

GTCTGAGTGACTTGTG

CACAAGTC

7ATTACCCA

CACTGTAGCTACAGTG

8GACTTGTG

GTGCACGA

TCGTGCAC

9TTCAATAG

TCCCACGA

TCGTGGGA

10GTTTGCTC

ACCAACAG

CTGTTGGT11ACCGGAGTAAGGAAGG

CCTTCCTT12CTTGACGAGCACACAATTGTGTGC

13

TGTTCGCC

AGGTAGGATCCTACCT14ACAAGGCATCGCGCAATTGCGCGA

15CCTGTCAAATGGCTGTACAGCCAT

16

CCATCCGC

AAGGCGTATACGCCTT

17ATGGCTGT

CCTGTCAATTGACAGG18

AAGGCGTACCATCCGCGCGGATGG

19

AGGTAGGATGTTCGCCGGCGAACA

20TCGCGCAAACAAGGCATGCCTTGT21

AAGGAAGG

ACCGGAGTACTCCGGT22GCACACAACTTGACGATCGTCAAG

23

TCCCACGATTCAATAG

CTATTGAA24

ACCAACAG

GTTTGCTCGAGCAAAC

19

96 Reaction Kit (NEB #E7120L)
For multiplexing < 96 samples use Table 3.3 for some valid index combinations.
Table 3.3.
PLEX

WELL POSITION<4

Not recommended
A6, B6, C6, and D6
A12, B12, C12, and D12
B6, C6, D6, and E6

4

B12, C12, D12, and E12
C1, D1, E1, and F1
C7, D7, E7, and F7
E4, F4, G4, and H4
E10, F10, G10, H10
A1, B1, C1, D1, E1
A6, B6, C6, D6, E6
A7, B7, C7, D7, E7
A12, B12, C12, D12, E12
B1, C1, D1, E1, F1
B6, C6, D6, E6, F6
B7, C7, D7, E7, F7

5

B12, C12, D12, E12, F12
C1, D1, E1, F1, G1
C2, D2, E2, F2, G2
C4, D4, E4, F4, G4
C7, D7, E7, F7, G7
C8, D8, E8, F8, G8
C10, D10, E10, F10, G10
D4, E4, F4, G4, H4
D10, E10, F10, G10, H10

6-7

Any 5 plex plus 1-2 adjacent wellsfrom the same column

8

Any column

20

* Forward Strand Workflow for the following instruments: NovaSeq 6000 with v1.0 reagents kits, MiniSeq with rapid reagent kits, MiSeq®,
HiSeq® 2000/2500 (pair-end flow cell), HiSeq 3000/4000 (single-read flow cell).
* Reverse Complement Workflow for the following instruments: iSeq 100, MiniSeq with standard reagent kits, NextSeq Systems,
NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/5000 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell).
Note: Please see Illumina Document “Indexed Sequencing Overview” document number 15057455
The index primer sequences, for different Illumina sequencer input sheets are indicated in Table 3.4.
Table 3.4 Index Sequences (Color coded based on HiSeq/MiSeq guidelines)
WELL
POSITIONEXPECTED i7 INDEX READ

A1
B1
C1
D1
E1
F1
G1
H1
A2
B2
C2
D2
E2
F2
G2
H2
A3
B3
C3
D3
E3
F3
G3
H3
A4
B4
C4
D4
E4
F4
G4
H4
A5
B5
C5
D5
E5
F5
G5
H5

TTACCGACTCGTCTGATTCCAGGTTACGGTCTAAGACCGT
CAGGTTCATAGGAGCTTACTCCAG
AGTGACCT
AGCCTATC
TCATCTCC
CCAGTATC
TTGCGAGAGAACGAAGCGAATTGCGGAAGAGATCGGATTCCTGTACCAGAGAGTACTCTACGCAGCAATTCCCTCAGAAG
GTCCTAAG
GCGTTAGACAAGGTACAGACCTTG
GTCGTTACGTAACCGAGAATCCGTCATGAGCA
CTTAGGACATCTGACC
TCCTCATGAGGATAGCGGAGGAATGACGTCAT
CCGCTTAAGACGAACTTCCACGTTAACCAGAGEXPECTED i5 INDEX READ
Forward Strand Workflow*

Reverse Complement Workflow*

CGAATACG
GTCCTTGACAGTGCTT
TCCATTGCGTCGATTGATAACGCC
GCCTTAACGGTATAGG
TCTAGGAGTGCGTAAC
CTTGCTAGAGCGAGATTATGGCACGAATCACCGTAAGGTGCGAGAGAACGCAACTA
CACAGACTTGGAAGCACAATAGCC
CTCGAACAGGCAAGTTAGCTACCACAGCATACCGTATCTCTTACGTGCAGCTAAGCAAGACACC
CAACTCCAGATCTTGCCTTCACTG
CTCGACTTGTACACCTCCAAGGTTGAACGGTT
CCAGTTGAGTCATCGTCAATGCGA
GGTTGAAC
CTTCGGTTCGTATTCG
TCAAGGACAAGCACTG
GCAATGGACAATCGACGGCGTTATGTTAAGGC
CCTATACC
CTCCTAGAGTTACGCACTAGCAAG
ATCTCGCTGTGCCATAGGTGATTCCACCTTACTTCTCTCG
TAGTTGCG
AGTCTGTG
TGCTTCCAGGCTATTGTGTTCGAG
AACTTGCCTGGTAGCTGTATGCTGGAGATACGGCACGTAAGCTTAGCT
GGTGTCTTTGGAGTTGGCAAGATCCAGTGAAG
AAGTCGAG
AGGTGTACAACCTTGG
AACCGTTCTCAACTGGACGATGACTCGCATTGGTTCAACC
AACCGAAG

21

WELL
POSITIONEXPECTED i7 INDEX READ

A6GTCAGTCAEXPECTED i5 INDEX READ
Forward Strand Workflow*

Reverse Complement Workflow*

CGGCATTATAATGCCG

B6

CCTTCCATCACGCAATATTGCGTGC6AGGAACACGGAATGTCGACATTCC

D6

CTTACAGCTGGTGAAG

CTTCACCA

E6

TACCTGCAGGACATCATGATGTCCF6

AGACGCTA

GGTGTACATGTACACCG6CAACACAGGATAGCCATGGCTATCH6

GTACCACACCACAACATGTTGTGGA7CGAATACGTTACCGACGTCGGTAA

B7

GTCCTTGATCGTCTGATCAGACGAC7

CAGTGCTT

TTCCAGGT

ACCTGGAA

D7

TCCATTGCTACGGTCTAGACCGTAE7GTCGATTGAAGACCGT

ACGGTCTTF7ATAACGCC

CAGGTTCATGAACCTG

G7

GCCTTAACTAGGAGCTAGCTCCTA

H7

GGTATAGGTACTCCAGCTGGAGTAA8TCTAGGAGAGTGACCTAGGTCACT

B8TGCGTAACAGCCTATCGATAGGCTC8CTTGCTAGTCATCTCCGGAGATGAD8AGCGAGATCCAGTATCGATACTGG

E8

TATGGCACTTGCGAGATCTCGCAAF8GAATCACCGAACGAAG

CTTCGTTCG8GTAAGGTGCGAATTGCGCAATTCGH8CGAGAGAAGGAAGAGATCTCTTCCA9CGCAACTATCGGATTCGAATCCGA

B9

CACAGACTCTGTACCATGGTACAGC9TGGAAGCAGAGAGTACGTACTCTC

D9CAATAGCC

TCTACGCATGCGTAGA

E9CTCGAACAGCAATTCCGGAATTGCF9GGCAAGTTCTCAGAAG

CTTCTGAG

G9AGCTACCAGTCCTAAG

CTTAGGACH9CAGCATACGCGTTAGATCTAACGC

A10CGTATCTCCAAGGTACGTACCTTG

B10

TTACGTGCAGACCTTG

CAAGGTCTC10AGCTAAGC

GTCGTTACGTAACGACD10AAGACACCGTAACCGATCGGTTAC

E10

CAACTCCAGAATCCGTACGGATTCF10GATCTTGCCATGAGCATGCTCATGG10CTTCACTG

CTTAGGACGTCCTAAG

H10

CTCGACTT

ATCTGACCGGTCAGAT22WELL
POSITIONEXPECTED i7 INDEX READ

A11

EXPECTED i5 INDEX READ
Forward Strand Workflow*

Reverse Complement Workflow*

GTACACCT

TCCTCATGCATGAGGA

B11CCAAGGTTAGGATAGCGCTATCCTC11GAACGGTTGGAGGAATATTCCTCC

D11

CCAGTTGAGACGTCATATGACGTC

E11

GTCATCGT

CCGCTTAATTAAGCGG

F11

CAATGCGAGACGAACTAGTTCGTCG11

GGTTGAAC

TCCACGTTAACGTGGA

H11CTTCGGTTAACCAGAG

CTCTGGTTA12CGGCATTAGTCAGTCATGACTGACB12CACGCAATCCTTCCATATGGAAGGC12GGAATGTCAGGAACAC

GTGTTCCT

D12TGGTGAAG

CTTACAGCGCTGTAAGE12

GGACATCATACCTGCATGCAGGTAF12GGTGTACAAGACGCTATAGCGTCTG12GATAGCCACAACACAG

CTGTGTTGH12CCACAACAGTACCACATGTGGTAC

Kit Components
NEB #E7120S Table of Components
NEB #PRODUCTVOLUME

E7122A

Control DNA CpG methylated pUC19

0.024 ml

E7123A

Control DNA Unmethylated Lambda

0.024 ml

E7646A

NEBNext Ultra II End Prep Enzyme Mix

0.168 ml

E7647A

NEBNext Ultra II End Prep Reaction Buffer

0.078 ml

E7374A

NEBNext Ligation Enhancer

0.024 ml

E7648A

NEBNext Ultra II Ligation Master Mix

0.72 ml

E7137A

NEBNext Sample Purification Beads

8.6 ml

E7124A

Elution Buffer

2.1 ml

E7126A

TET2 Reaction Buffer

0.3 ml

E7127A

TET2 Reaction Buffer Supplement (x 3)

powder

E7128A

Oxidation Supplement

0.024 ml

E7139AA

DTT

E7129A

Oxidation Enhancer

0.024 ml

E7130A

TET2

0.096 mlE7131A

Fe(II) Solution

0.024 ml

E7132A

Stop Reagent

0.024 ml

E7133A

APOBEC0.024 ml

E7134A

APOBEC Reaction Buffer

0.24 ml

E7135A

BSA

0.024 ml

E7136A

NEBNext Q5U Master Mix

E7165A

NEBNext EM-seq Adaptor

0.06 ml

E7141A

EM-seq Index Primer 1

0.005 ml

E7142A

EM-seq Index Primer 2

0.005 ml

E7143A

EM-seq Index Primer 3

0.005 ml

E7144A

EM-seq Index Primer 4

0.005 ml

E7145A

EM-seq Index Primer 5

0.005 ml

0.5 ml

0.6 ml23

E7146A

EM-seq Index Primer 6

0.005 ml

E7147A

EM-seq Index Primer 7

0.005 ml

E7148A

EM-seq Index Primer 8

0.005 ml

E7149A

EM-seq Index Primer 9

0.005 ml

E7150A

EM-seq Index Primer 10

0.005 ml

E7151A

EM-seq Index Primer 11

0.005 ml

E7152A

EM-seq Index Primer 12

0.005 ml

E7153A

EM-seq Index Primer 13

0.005 ml

E7154A

EM-seq Index Primer 14

0.005 ml

E7155A

EM-seq Index Primer 15

0.005 ml

E7156A

EM-seq Index Primer 16

0.005 ml

E7157A

EM-seq Index Primer 17

0.005 ml

E7158A

EM-seq Index Primer 18

0.005 ml

E7159A

EM-seq Index Primer 19

0.005 ml

E7160A

EM-seq Index Primer 20

0.005 mlE7161A

EM-seq Index Primer 21

0.005 ml

E7162A

EM-seq Index Primer 22

0.005 ml

E7163A

EM-seq Index Primer 23

0.005 ml

E7164A

EM-seq Index Primer 24

0.005 ml

NEB #E7120L Table of Components
NEB #PRODUCTVOLUME

E7122AA

Control DNA CpG methylated pUC19

0.096 ml

E7123AA

Control DNA Unmethylated Lambda

0.096 ml

E7646AA

NEBNext Ultra II End Prep Enzyme Mix

0.288 ml

E7647AA

NEBNext Ultra II End Prep Reaction Buffer

0.672 ml

E7374AA

NEBNext Ligation Enhancer

0.096 ml

E7648AA

NEBNext Ultra II Ligation Master Mix

2.88 ml

E7137AA

NEBNext Sample Purification Beads

34.6 ml

E7124AA

Elution Buffer

E7126AATET2 Reaction Buffer

1.2 ml

E7127AATET2 Reaction Buffer Supplement (x 3)

powder

E7128AA

Oxidation Supplement

0.096 ml

E7139AA

DTT

E7129AA

Oxidation Enhancer

0.096 ml

E7130AA

TET2

0.384 mlE7131AA

Fe(II) Solution

0.096 ml

E7132AA

Stop Reagent

0.096 ml

E7133AA

APOBEC

0.096 ml

E7134AA

APOBEC Reaction Buffer

0.96 ml

E7135AA

BSA

0.096 ml

E7136AA

NEBNext Q5U Master Mix

E7165AA

NEBNext EM-seq Adaptor

E7166A

NEBNext 96 Unique Dual Index Primer Pairs Plate

9 ml

0.5 ml2.4 ml0.24 ml
0.005 ml x 96

24

CheckList (Section 1)
1.1 DNA Preparation
1.1.1.Combine DNA and control DNA
Sequencing on MiSeq before NovaSeq, HiSeq or NextSeq (Check 2-4 M paired end reads per EM-seq Library)[ _ ] 10–200 ng sample DNA[ _ ] 1 µl of 0.1 ng/µl pUC19 control DNA (lilac)[ _ ] 1 µl of 2 ng/µl unmethylated lambda DNA (lilac)[ _ ] Add 0.1X TE pH 8.0 to 50 µl
For direct sequencing on NovaSeq, HiSeq or NextSeq[ _ ] 10–200 ng sample DNA[ _ ] 1 µl of pUC19 DNA (lilac) diluted 100X to 0.001 ng/µl pUC19[ _ ] 1 µl of unmethylated lambda DNA (lilac) diluted 100X to 0.02 ng/µl[ _ ] Add 0.1X TE pH 8.0 to 50 µl
1.1.2.Shear DNA[ _ ] Shear to 240–290 bp, use preferred instrument.
[ _ ] Transfer the 50 µl of sheared material directly to a PCR strip tube to begin library construction.
1.2. End Prep of Sheared DNA
Add End Prep Reagents to sample (50 µl):
[ _ ] 7 µl NEBNext Ultra II End Prep Reaction Buffer (green)[ _ ] 3 µl NEBNext Ultra II End Prep Enzyme Mix (green)[ _ ] Vortex or pipette mix 10 times with pipette, quick spin[ _ ] Incubate in thermal cycler
30 minutes 20°C
30 minutes 65°C
Hold at 4°C
1.3. Ligation of EM-seq Adaptor
Add Ligation Reagents to End Repaired DNA[ _ ] 2.5 µl NEBNext EM-seq adaptor (red)[ _ ] 1 µl NEBNext Ligation Enhancer (red)[ _ ] 30 µl NEBNext Ultra II Ligation Master Mix (red)[ _ ] Vortex or pipette mix 10 times with pipette, quick spin[ _ ] Incubate in thermal cycler
15 minutes 20°C (heated lid off)
Hold at 4°C
1.4. Clean-Up of Adaptor Ligated DNA[ _ ] Vortex beads[ _ ] Add 110 µl of resuspended NEBNext Sample Purification Beads to each sample and mix by pipetting 10 times[ _ ] Incubate 5 min[ _ ] Place tubes on magnet for 5 min[ _ ] Remove and discard the supernatant, while keeping the sample on the magnet[ _ ] On magnet add 200 µl 80% ethanol, wait 30 seconds and then remove and discard the ethanol wash
[ _ ] Repeat the 80% ethanol wash
[ _ ] Airdry the beads for up to 2 min while on magnet[ _ ] Remove the samples from the magnet and resuspend in 29 µl of Elution Buffer (white)[ _ ] Place back on the magnet, wait until the supernatant clears and transfer 28 µl of sample to fresh PCR tubes

25

1.5. Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines[ _ ] Reconstitute the TET2 Reaction Buffer Supplement (yellow) using TET2 Reaction Buffer (yellow)
24 Reaction kit: 1 tube TET2 Reaction Buffer Supplement (yellow) & 100 µl TET2 Reaction Buffer (yellow)
96 Reaction kit: 1 tube TET2 Reaction Buffer Supplement (yellow) & 400 µl TET2 Reaction Buffer (yellow)
Once reconstituted the Tet2 Reaction Buffer should be used within 4 months[ _ ] Add Oxidation Reagents to 28 µl Adaptor Ligated DNA[ _ ] 10 µl Reconstituted TET2 Reaction Buffer (yellow)[ _ ] 1 µl Oxidation Supplement (yellow)[ _ ] 1 µl DTT (yellow)[ _ ] 1 µl Oxidation Enhancer (yellow)[ _ ] 4 µl TET2 (yellow)[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Make diluted Fe(II) Solution.
Add 1 µl 500 mM Fe(II) Solution (yellow) to 1,249 µl of water. Use immediately, do not store.
[ _ ] Add 5 µl Diluted Fe(II) Solution to sample[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly, then incubate in a thermal cycler
60 minutes 37°C
Hold at 4°C[ _ ] Add 1 µl Stop Reagent (yellow)[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Incubate in a thermal cycler
30 minutes 37°C
Hold at 4°C
1.6. Clean-Up of TET2 converted DNA[ _ ] Vortex beads[ _ ] Add 90 µl of resuspended NEBNext Sample Purification Beads to each sample and mix by pipetting 10 times[ _ ] Incubate 5 min[ _ ] Place tubes on magnet for 5 min[ _ ] Remove and discard the supernatant, while keeping the sample on the magnet[ _ ] On magnet add 200 µl 80% ethanol, wait 30 seconds and then remove and discard the ethanol wash
[ _ ] Repeat the 80% ethanol wash
[ _ ] Airdry the beads for up to 2 min while on magnet[ _ ] Remove the samples from the magnet and resuspend in 17 µl of Elution Buffer (white)[ _ ] Place back on the magnet, wait until the supernatant clears and transfer 16 µl of sample to fresh PCR tubes
1.7. Denaturation of DNA
Use either Formamide (A) or Sodium Hydroxide (B)A. Formamide Denaturation (Recommended)[ _ ] Pre-heat thermal cycler to 85°C
[ _ ] Add 4 µl Formamide to the 16 µl oxidized DNA[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Incubate in a thermal cycler
85°C for 10 min
Immediately, place on ice[ _ ] Proceed immediately into Section 1.8
B. Sodium Hydroxide Denaturation (Optional: Formamide is preferred)[ _ ] Prepare freshly diluted 0.1 N NaOH
[ _ ] Pre-heat thermal cycler to 50°C
[ _ ] Add 4 µl 0.1 N NaOH to the 16 µl oxidized DNA26

[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Incubate in a thermal cycler
50°C for 10 min
Immediately, place on ice[ _ ] Proceed immediately into Section 1.8
1.8. Deamination of Cytosines
Add Deamination Reagents to 20 µl denatured DNA on ice[ _ ] 68 µl Nuclease-free water[ _ ] 10 µl APOBEC Reaction Buffer (orange)[ _ ] 1 µl BSA (orange)[ _ ] 1 µl APOBEC (orange)[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Incubate in a thermal cycler
37°C for 3 hours
Hold at 4°C
1.9. Clean-Up of Deaminated DNA[ _ ] Vortex beads[ _ ] Add 100 µl of resuspended NEBNext Sample Purification Beads to each sample and mix by pipetting 10 times[ _ ] Incubate 5 min[ _ ] Place tubes on magnet for 5 min[ _ ] Remove and discard the supernatant, while keeping the sample on the magnet[ _ ] On magnet add 200 µl 80% ethanol, wait 30 seconds and then remove and discard the ethanol wash
[ _ ] Repeat the 80% ethanol wash
[ _ ] Airdry the beads for up to 90 sec while on magnet. Do not over-dry as beads become difficult to resuspend[ _ ] Remove the samples from the magnet and resuspend in 21 µl of Elution Buffer (white)[ _ ] Place back on the magnet, wait until the supernatant clears and transfer 20 µl of sample to fresh PCR tubes
1.10 PCR Amplification
Add Amplification Reagents to 20 µl deaminated DNA[ _ ] 5 µl NEBNext Unique Dual Index Primer Pairs[ _ ] 25 µl NEBNext Q5U Master Mix (blue)[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Amplify in thermal cycler
CYCLE STEP
Initial Denaturation
Denaturation
Annealing

TEMPERATURE
98°C
98°C
62°C

TIME
CYCLES
30 s
1
10 s
30 s
4–8*

Extension

65°C

60 s

Final Extension
Hold
Cycle recommendation:

65°C
4

5 min
∞

1

10 ng DNA: 8 cycles
50 ng DNA: 5–6 cycles
200 ng DNA: 4 cycles

27

1.11.Clean-Up of Amplified Libraries
Vortex beads[ _ ] Add 45 µl of resuspended NEBNext Sample Purification Beads to each sample and mix by pipetting 10 times[ _ ] Incubate 5 min[ _ ] Place tubes on magnet for 5 min[ _ ] Remove and discard the supernatant, while keeping the sample on the magnet[ _ ] On magnet add 200 µl 80% ethanol, wait 30 seconds and then remove and discard the ethanol wash
[ _ ] Repeat the 80% ethanol wash
[ _ ] Airdry the beads for up to 2 min while on magnet[ _ ] Remove the samples from the magnet and resuspend in 21 µl of Elution Buffer (white) or 10 mM Tris, 0.1 mM EDTA,
pH 8.0 (for long term storage)[ _ ] Place back on the magnet, wait until the supernatant clears and transfer 20 µl of sample to fresh PCR tubes
1.12 Library Quantification and Sequencing[ _ ] Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries[ _ ] Sequence using the preferred Illumina platform. 2 x 76 base or 2 x 100 base reads for standard sized libraries.

28

CheckList (Section 2)
2.1. DNA Preparation
2.1.1.Combine DNA and control DNA
Sequencing on MiSeq before NovaSeq, HiSeq or NextSeq (Check 2-4 M paired end reads per EM-seq Library)[ _ ] 10–200 ng sample DNA[ _ ] 1 µl of 0.1 ng/µl pUC19 control DNA (lilac)[ _ ] 1 µl of 2 ng/µl unmethylated lambda DNA (lilac)[ _ ] Add 0.1X TE pH 8.0 to 50 µl
For direct sequencing on NovaSeq, HiSeq or NextSeq[ _ ] 10–200 ng sample DNA[ _ ] 1 µl of pUC19 DNA (lilac) diluted 100X to 0.001 ng/µl pUC19[ _ ] 1 µl of unmethylated lambda DNA (lilac) diluted 100X to 0.02 ng/µl[ _ ] Add 0.1X TE pH 8.0 to 50 µl
2.1.2.Shear DNA[ _ ] Shear to 350–400 bp, use preferred instrument.
[ _ ] Transfer the 50 µl of sheared material directly to a PCR strip tube to begin library construction.
2.2. End Prep of Sheared DNA
Add End Prep Reagents to sample (50 µl):
[ _ ] 7 µl NEBNext Ultra II End Prep Reaction Buffer (green)[ _ ] 3 µl NEBNext Ultra II End Prep Enzyme Mix (green)[ _ ] Vortex or pipette mix 10 times with pipette, quick spin[ _ ] Incubate in thermal cycler
30 minutes 20°C
30 minutes 65°C
Hold at 4°C
2.3. Ligation of EM-seq Adaptor
Add Ligation Reagents to End Repaired DNA[ _ ] 2.5 µl NEBNext EM-seq adaptor (red)[ _ ] 1 µl NEBNext Ligation Enhancer (red)[ _ ] 30 µl NEBNext Ultra II Ligation Master Mix (red)[ _ ] Vortex or pipette mix 10 times with pipette, quick spin[ _ ] Incubate in thermal cycler
15 minutes 20°C (heated lid off)
Hold at 4°C
2.4. Clean-Up of Adaptor Ligated DNA[ _ ] Vortex beads[ _ ] Add 110 µl of resuspended NEBNext Sample Purification Beads to each sample and mix by pipetting 10 times[ _ ] Incubate 5 min[ _ ] Place tubes on magnet for 5 min[ _ ] Remove and discard the supernatant, while keeping the sample on the magnet[ _ ] On magnet add 200 µl 80% ethanol, wait 30 seconds and then remove and discard the ethanol wash
[ _ ] Repeat the 80% ethanol wash
[ _ ] Airdry the beads for up to 2 min while on magnet[ _ ] Remove the samples from the magnet and resuspend in 29 µl of Elution Buffer (white)[ _ ] Place back on the magnet, wait until the supernatant clears and transfer 28 µl of sample to fresh PCR tubes

29

2.5. Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines[ _ ] Reconstitute the TET2 Reaction Buffer Supplement • (yellow) using TET2 Reaction Buffer • (yellow)
24 Reaction kit: 1 tube TET2 Reaction Buffer Supplement • (yellow) & 100 µl TET2 Reaction Buffer • (yellow)
96 Reaction kit: 1 tube TET2 Reaction Buffer Supplement • (yellow) & 400 µl TET2 Reaction Buffer • (yellow)
Once reconstituted the Tet2 Reaction Buffer should be used within 4 months[ _ ] Add Oxidation Reagents to 28 µl Adaptor Ligated DNA[ _ ] 10 µl Reconstituted TET2 Reaction Buffer • (yellow)[ _ ] 1 µl Oxidation Supplement • (yellow)[ _ ] 1 µl DTT • (yellow)[ _ ] 1 µl Oxidation Enhancer • (yellow)[ _ ] 4 µl TET2 • (yellow)[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Make diluted Fe(II) Solution.
Add 1 µl 500 mM Fe(II) Solution • (yellow) to 1,249 µl of water. Use immediately, do not store.
[ _ ] Add 5 µl Diluted Fe(II) Solution to sample.
[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly, then incubate in a thermal cycler
60 minutes 37°C
Hold at 4°C[ _ ] Add 1 µl Stop Reagent • (yellow)[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Incubate in a thermal cycler
30 minutes 37°C
Hold at 4°C
2.6. Clean-Up of TET2 converted DNA[ _ ] Vortex beads[ _ ] Add 90 µl of resuspended NEBNext Sample Purification Beads to each sample and mix by pipetting 10 times[ _ ] Incubate 5 min[ _ ] Place tubes on magnet for 5 min[ _ ] Remove and discard the supernatant, while keeping the sample on the magnet[ _ ] On magnet add 200 µl 80% ethanol, wait 30 seconds and then remove and discard the ethanol wash
[ _ ] Repeat the 80% ethanol wash
[ _ ] Airdry the beads for up to 2 min while on magnet[ _ ] Remove the samples from the magnet and resuspend in 17 µl of Elution Buffer (white)[ _ ] Place back on the magnet, wait until the supernatant clears and transfer 16 µl of sample to fresh PCR tubes
2.7. Denaturation of DNA
Use either Formamide (A) or Sodium Hydroxide (B)A. Formamide Denaturation (Recommended)[ _ ] Pre-heat thermal cycler to 85°C
[ _ ] Add 4 µl Formamide to the 16 µl oxidized DNA[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Incubate in a thermal cycler
85°C for 10 min
Immediately, place on ice[ _ ] Proceed immediately into Section 2.8
B. Sodium Hydroxide Denaturation (Optional: Formamide is preferred)[ _ ] Prepare freshly diluted 0.1 N NaOH
[ _ ] Pre-heat thermal cycler to 50°C
[ _ ] Add 4 µl 0.1 N NaOH to the 16 µl oxidized DNA30

[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Incubate in a thermal cycler
50°C for 10 min
Immediately, place on ice[ _ ] Proceed immediately into Section 2.8
2.8. Deamination of Cytosines
Add Deamination Reagents to 20 µl denatured DNA on ice[ _ ] 68 µl Nuclease-free water[ _ ] 10 µl APOBEC Reaction Buffer (orange)[ _ ] 1 µl BSA (orange)[ _ ] 1 µl APOBEC (orange)[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Incubate in a thermal cycler
37°C for 3 hours
Hold at 4°C
2.9. Clean-Up of Deaminated DNA[ _ ] Vortex beads[ _ ] Add 100 µl of resuspended NEBNext Sample Purification Beads to each sample and mix by pipetting 10 times[ _ ] Incubate 5 min[ _ ] Place tubes on magnet for 5 min[ _ ] Remove and discard the supernatant, while keeping the sample on the magnet[ _ ] On magnet add 200 µl 80% ethanol, wait 30 seconds and then remove and discard the ethanol wash
[ _ ] Repeat the 80% ethanol wash
[ _ ] Airdry the beads for up to 90 sec while on magnet. Do not over-dry as beads become difficult to resuspend[ _ ] Remove the samples from the magnet and resuspend in 21 µl of Elution Buffer (white)[ _ ] Place back on the magnet, wait until the supernatant clears and transfer 20 µl of sample to fresh PCR tubes
2.10. PCR Amplification
Add Amplification Reagents to 20 µl deaminated DNA[ _ ] 5 µl NEBNext Unique Dual Index Primer Pairs[ _ ] 25 µl NEBNext Q5U Master Mix (blue)[ _ ] Mix by vortexing or pipette mix 10 times, centrifuge briefly[ _ ] Amplify in thermal cycler
CYCLE STEPTEMPERATURE

TIME

CYCLES

Initial Denaturation

98°C

30 s

1

Denaturation

98°C

10 s

Annealing

62°C

30 s

Extension

65°C

60 s

Final Extension

65°C

5 min

Hold

4

∞

4–8*

1

Cycle recommendation:
10 ng DNA: 8 cycles
50 ng DNA: 5–6 cycles
200 ng DNA: 4 cycles

31

2.11. Clean-Up of Amplified Libraries
Vortex beads[ _ ] Add 90 µl of water to each sample[ _ ] Add 91 µl of resuspended NEBNext Sample Purification Beads to each sample and mix by pipetting 10 times[ _ ] Incubate 5 min[ _ ] Place tubes on magnet for 5 min[ _ ] Remove and discard the supernatant, while keeping the sample on the magnet[ _ ] On magnet add 200 µl 80% ethanol, wait 30 seconds and then remove and discard the ethanol wash
[ _ ] Repeat the 80% ethanol wash
[ _ ] Airdry the beads for up to 2 min while on magnet[ _ ] Remove the samples from the magnet and resuspend in 21 µl of Elution Buffer (white) or 10 mM Tris, 0.1 mM EDTA,
pH 8.0 (for long term storage)[ _ ] Place back on the magnet, wait until the supernatant clears and transfer 20 µl of sample to fresh PCR tubes
2.12. Library Quantification and Sequencing[ _ ] Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries[ _ ] Sequence using the preferred Illumina platform. 2 x 100 base or 2 x 150 base reads for large sized libraries.

32

Revision History
REVISION #DESCRIPTION

DATE

1.0

N/A

4/19

2.0

Updated items in Checklist (Steps 1.5 and 1.10) also (Steps 2.5
and 2.10).

7/19

3.0

Correct step numbers in Section 2. Steps 2.7A and 2.7B.

8/19

4.0

Add DTT to kit and update protocol to include DTT.

2/20

5.0

Change Elution Buffer amount in Step 2.4.9 to 29 µl and in
Step 2.4.11 change 29 µl supernatant to 28 µl. Step 1.5.2 and2.5.2 updated the component description.

2/20

6.0

New format applied.

3/20

7.0

Protocol updates, updated table formatting,updated legal footer

4/23

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.
This product 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. See
www.neb.com/trademarks. The use of this product may require you to obtain additional third-party intellectual property rights for certain applications. For more information,please email busdev@neb.com.
This product is covered by one or more patents owned by New England Biolabs, Inc. and is intended for research purposes. It is not intended for use in therapeutic or diagnostic
uses in humans or animals. Additionally, 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 is licensed from University of Newcastle Upon Tyne under EPO Patent No. 1463809B1 and Australian Patent No. AU2003226543.
This product is licensed 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 for use
only in: (a) standard (not real-time) PCR in the research field only, but not real time PCR or digital PCR; (b) real-time PCR for use as a library preparation quantitation tool in
Next Generation Sequencing workflows; (c) any in-vitro diagnostics applications, except for applications using real-time PCR or digital PCR; and (d) any non-PCR
applications in DNA sequencing, isothermal amplification, and the production of synthetic DNA.
Notice to Purchaser: Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc.
(See Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,874,557; and 5,693,502). New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the aptamerbased NEBNext® Q5U™ Master Mix for RESEARCH PURPOSES ONLY. Commercial use of the aptamer-based NEBNext Q5U Master Mix requires a license from New
England Biolabs, Inc. Please contact busdev@neb.com for more information.
B CORPORATION® is a registered trademark of B Lab IP, LLC, Inc.
ILLUMINA®, HISEQ®, MISEQ®, NOVASEQ® and NEXTSEQ® and MINISEQ® are registered trademarks of Illumina, Inc.
COVARIS® is a registered trademark of Covaris, Inc.
BIOANALYZER® and TAPESTATION® are registered trademarks of Agilent Technologies© Copyright 2023, New England Biolabs, Inc.; all rights reserved

33