TITLE: Protocol for Use with Large Insert Libraries (470–520 bp) (NEB #E7120)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to construct Large Insert libraries from start to finish using NEBNext reagents. </div><div class = "text-block"><span>The corresponding NEB manual is here: </span><a href="https://www.neb.com/-/media/nebus/files/manuals/manuale7120.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.neb.com/-/media/nebus/files/manuals/manuale7120.pdf</span></a><span> and this protocol relates to section 2.</span></div><div class = "text-block"><span style = "font-weight:bold;">Overview</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/f7d8df35bc17c0a1a76c218d6aa1aa0753d64d15ea2475e12d694a432b3d2305/cqdjba7dp.png" /></div><div class = "text-block">The Enzymatic Methyl-seq kit (EM-seq) for Illumina contains all the components needed to make libraries that are enzymatically modified to detect 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC). </div><div class = "text-block">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 cytosines from 5mC and 5hmC. Finally, libraries are PCR amplified before sequencing.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/f7d8df35bc17c0a1a76c218d6aa1aa0753d64d15ea2475e12d694a432b3d2305/cqdhba7dp.png" /></div><div class = "text-block">Figure 2 shows a comparison of the sodium bisulfite and EM-seq methods. Sodium bisulfite treatment of DNA results in the deamination of cytosines into uracils, however the modified forms of cytosine (5mC and 5hmC) are not deaminated. Therefore, the preference of bisulfite 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 the appropriate methylation status can be assessed.</div><div class = "text-block">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) Þ5-hydroxymethylcytosine (5hmC) Þ 5-formylcytosine (5fC) Þ 5- carboxycytosine (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 and 5ghmC. 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.</div><div class = "text-block">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. </div><div class = "text-block">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. </div><div class = "text-block">For larger volume requirements, customized and bulk packaging is available by purchasing through the Custom Solutions department at NEB. Please contact Custom@neb.com for further information.</div></div>

[STEPS]
?. [DNA Preparation (Section 2.1)]
DNA PreparationCombine 10 ng-200 ng of genomic DNA with control DNA, CpG methylated pUC19 (lilac) and unmethylated lambda DNA (lilac) in  made up with 0.1X TE . The amount of control DNA added is dependent on the number of reads required.
Starting materials is 10 ng-200 ng DNA
50 µl
If checking library quality on a MiSeq®(2–4 M reads per library) prior to deep sequencing on NovaSeq®, HiSeq®or NextSeq® (100–500 M reads per library) then the amount of controls spiked to the sample DNA is higher than what is required for direct deep sequencing. Having higher ng of control DNA for samples that are sequenced on a MiSeq ensures that there are enough control reads to accurately call cytosine conversion. We recommend this for users who are inexperienced with next generation sequencing library preparation. For libraries sequenced to a depth of 2–4 M paired end reads, approximately 5,000 x 76 base paired end reads of unmethylated lambda and 500 x 76 base paired end reads of CpG methylated pUC19 are needed to give enough reads for accurate conversion estimates. If these same libraries are sequenced to a higher depth of 200–400 M reads per library, then the number of reads associated with the controls would be in vast excess, 500,000 for unmethylated lambda and 50,000 for pUC19.Recommended Control Inputs:Pre-sequencing on MiSeq prior to deep sequencing on NovaSeq, HiSeq or NextSeq: spike in 1 μl of 0.1 ng/μl pUC19 control DNA (lilac) and 1 μl of 2 ng/μl unmethylated lambda DNA (lilac) per 10–200 ng sample DNA.Direct Sequencing on NovaSeq, HiSeq or NextSeq: Dilute the pUC19 (lilac) and the unmethylated lambda control (lilac) 1:100 using 0.1X TE, pH 8.0. Spike in 1 μl diluted pUC19 (0.001 ng) control DNA and 1 μl diluted unmethylated lambda DNA (0.02 ng) per 10–200 ng sample DNA
?. [DNA Preparation (Section 2.1)]
Shearing DNATransfer the  to a new PCR tube for End Prep.
The combined   and control DNAs are fragmented to an average insert size of 350–400 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.
[sheared DNA]
DNA does not need to be cleaned up or size selected before End Prep.
?. [End Prep of Sheared DNA (Section 2.2)]
, mix the following components in a sterile nuclease-free PCR tube:  AB1COMPONENTVOLUME2Fragmented DNA50 µl3(green) NEBNext Ultra II End Prep Reaction Buffer7 µl4(green) NEBNext Ultra II End Prep Enzyme Mix3 µl5Total Volume60 µl
on ice
AB1COMPONENTVOLUME2Fragmented DNA50 µl3(green) NEBNext Ultra II End Prep Reaction Buffer7 µl4(green) NEBNext Ultra II End Prep Enzyme Mix3 µl5Total Volume60 µl
?. [End Prep of Sheared DNA (Section 2.2)]
Set a  or  pipette to  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.
100 µl
200 µl
50 µl
It is important to mix well. The presence of a small amount of bubbles will not interfere with the performance.
?. [End Prep of Sheared DNA (Section 2.2)]
Place in a thermocycler with the heated lid set to ≥  or on, and run the following program: at  at Hold at
75 °C
20 °C
65 °C
4 °C
?. [Ligation of EM-seq Adaptor (Section 2.3)]
, add the following components directly to the  and mix well:  AB1COMPONENTVOLUME2(red) NEBNext EM-seq Adaptor2.5 µl3(red) NEBNext Ligation Enhancer1 µl4(red) NEBNext Ultra II Ligation Master Mix30 µl5Total Volume93.5 µl
on ice
[End Prep reaction mixture]
AB1COMPONENTVOLUME2(red) NEBNext EM-seq Adaptor2.5 µl3(red) NEBNext Ligation Enhancer1 µl4(red) NEBNext Ultra II Ligation Master Mix30 µl5Total Volume93.5 µl
Ligation Enhancer and Ligation Master Mix can be mixed ahead of time and is stable for at least 8 hours at  . We do not recommend adding adaptor to a premix in the adaptor ligation step. Premix adaptor and sample and then add the other ligation reagents.
?. [Ligation of EM-seq Adaptor (Section 2.3)]
Set a  or  pipette to  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.
100 µl
200 µl
80 µl
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 interfere with performance.
?. [Ligation of EM-seq Adaptor (Section 2.3)]
Incubate at  for  in a thermocycler with the heated lid off.
20 °C
SAFE STOPPING POINT: Samples can be stored overnight at  .
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Vortex Sample Purification Beads to resuspend.
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[of resuspended NEBNext Sample Purification Beads]
Be careful to expel all of the liquid out of the tip during the last mix.
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Incubate samples on bench top for at least  at .
0 Room temperature
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
After , or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targetsCAUTION: DO NOT discard the beads
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Add   to the tubes while in the magnetic stand.
[of 80% freshly prepared ethanol]
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
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.
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Air dry the beads for up to 2 minutes while the tubes are on the magentic 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 brown and start to crack they are too dry.
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding  (white).
[of Elution Buffer]
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Mix well by pipetting up and down 10 times. Incubate for at least  at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Place the tube on the magnetic stand. After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at  .
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 2.5)]
Prepare TET2 Buffer. Use option A if you have E7120S/E7120G (24 Reactions/G size) and option B if you have E7120L (96 reactions).Option A: E7120S/E7120GAdd  to one tube of TET2 Reaction Buffer Supplement and mix well. Write date on tube.Option B: E7120LAdd   to one tube of TET2 Reaction Buffer Supplement and mix well. Write date on tube.Option B: E7120LAdd  to one tube of TET2 Reaction Buffer Supplement and mix well.
The TET2 Reaction Buffer Supplement is a powder. Centrifuge before use to ensure it is at the bottom of the tube.
[of TET2 Reaction Buffer]
[of TET2 Reaction Buffer]
The reconstituted buffer should be stored at   and discarded after 4 months.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 2.5)]
, add the following components directly to the  (from step 20).  AB1COMPONENTVOLUME2(yellow) TET2 Reaction Buffer (TET2 Reaction Buffer plus reconstituted TET2 Reaction Buffer Supplement)10 µl3(yellow) Oxidation Supplement1 µl4(yellow) DTT1 µl5(yellow) Oxidation Enhancer1  µl6(yellow) TET24 µlMix thoroughly by vortexing, centrifuge briefly.
on ice
[EM-seq adaptor ligated DNA]
AB1COMPONENTVOLUME2(yellow) TET2 Reaction Buffer (TET2 Reaction Buffer plus reconstituted TET2 Reaction Buffer Supplement)10 µl3(yellow) Oxidation Supplement1 µl4(yellow) DTT1 µl5(yellow) Oxidation Enhancer1  µl6(yellow) TET24 µl
For multiple reactions, a master mix of the reaction components can be prepared before addition to the sample DNA. 5mC/5hmC oxidation is initiated by the addition of the Fe(II) solution to the reaction in the next step.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 2.5)]
Dilute the  by adding  to .Combine Diluted Fe(II) Solution and EM-seq DNA with Oxidation Enzymes (from Step 22).  AB1COMPONENTVOLUME2EM-seq DNA (from step 22)45 μl3Diluted Fe(II) Solution (from step 23)5 μl4Total Volume50 μlMix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
[Fe(II) Solution (yellow)]
1 µl
[of water]
Use the solution immediately, do not store it. Discard after use.
AB1COMPONENTVOLUME2EM-seq DNA (from step 22)45 μl3Diluted Fe(II) Solution (from step 23)5 μl4Total Volume50 μl
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 2.5)]
Incubate at  for  in a thermocycler with the heated lid set to ≥   or on.
37 °C
45 °C
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 2.5)]
Transfer the samples to ice and add  (yellow).  AB1COMPONENTVOLUME2(yellow) Stop Reagent1 μl3Total Volume51 μlMix thoroughly by vortexing or by pipetting up and down at least 10 times and centrifuge briefly.
[of Stop Reagent]
AB1COMPONENTVOLUME2(yellow) Stop Reagent1 μl3Total Volume51 μl
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 2.5)]
Incubate at  for   then at  in the thermocycler with the heated lid set to ≥  or on.
37 °C
4 °C
45 °C
SAFE STOPPING POINT: Samples can be stored overnight at either   in the thermocycler or at   in the freezer.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Vortex Sample Purification Beads to resuspend.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[of resuspended NEBNext Sample Purification Beads]
Be careful to expel all of the liquid out of the tip during the last mix.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Incubate samples on bench top for at least  at .
0 Room temperature
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
After , or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targetsCAUTION: DO NOT discard the beads.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Add  to the tubes while in the magnetic stand. Incubate at  for  , then carefully remove and discard the supernatant.
[of 80% freshly prepared ethanol]
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Repeat the previous wash once for a total of two washes.
Be sure to remove all visible liquid after the second wash using a p10 pipette tip.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
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 brown and start to crack they are too dry.
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 brown and start to crack they are too dry.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding .
[of Elution Buffer (white)]
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Clean-Up of TET2 Converted DNA (Section 2.6)]
Place the tube on the magnetic stand. After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at -20°C.
?. [Denaturation of DNA (Section 2.7)]
Use option A for denaturing using Formamide and option B for denaturing using 0.1 N Sodium hydroxide.Option A: Formamide (Recommended) Pre-heat thermocycler to . Add  to the . Vortex to mix or by pipetting up and down at least 10 times, centrifuge briefly. Incubate at  for  in the pre-heated thermocycler with the heated lid on.Immediately place . Proceed immediately to the next section.Option B: Sodium Hydroxide (Optional, see FAQ about preparing NaOH) Prepare freshly diluted 0.1 N NaOH. Pre-heat thermocycler to   with the heated lid set to ≥   or on. Add   to the  . Vortex to mix or by pipetting up and down at least 10 times, centrifuge briefly.Incubate at   for   in the pre-heated thermocycler with the heated lid set to ≥   or on.Immediately place on ice.Proceed immediately to the next section.Option B: Sodium Hydroxide (Optional, see FAQ about preparing NaOH) Prepare freshly diluted 0.1 N NaOH. Pre-heat thermocycler to   with the heated lid set to ≥   or on. Add   to the  . Vortex to mix or by pipetting up and down at least 10 times, centrifuge briefly.Incubate at   for   in the pre-heated thermocycler with the heated lid set to ≥   or on.Immediately place on ice.Proceed immediately to the next section.Pre-heat thermocycler to  with the heated lid set to ≥  or on. Add  to the . Vortex to mix or by pipetting up and down at least 10 times, centrifuge briefly.Incubate at  for  in the pre-heated thermocycler with the heated lid set to ≥  or on.Immediately place on ice.Proceed immediately to the next section.
The DNA can be denatured using either Formamide or 0.1 N Sodium Hydroxide.
85 °C
[Formamide]
[of oxidized DNA]
85 °C
on ice
50 °C
60 °C
[0.1 N NaOH]
16 µl
50 °C
60 °C
?. [Deamination of Cytosines (Section 2.8)]
, add the following components to the .  AB1COMPONENTVOLUME2Nuclease-free water68 µl3(orange) APOBEC Reaction Buffer10 µl4(orange) BSA1 µl5(orange) APOBEC1 µl6Total Volume100 µl
on ice
[of denatured DNA]
AB1COMPONENTVOLUME2Nuclease-free water68 µl3(orange) APOBEC Reaction Buffer10 µl4(orange) BSA1 µl5(orange) APOBEC1 µl6Total Volume100 µl
For multiple reactions, a master mix of the reaction components can be prepared before addition to the denatured DNA.
?. [Deamination of Cytosines (Section 2.8)]
Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
?. [Deamination of Cytosines (Section 2.8)]
Incubate at  for , then at  in a thermocycler with the heated lid set to ≥ or on.
37 °C
4 °C
45 °C
SAFE STOPPING POINT: Samples can be stored overnight at either   in the thermocycler or at   in the freezer.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
Vortex Sample Purification Beads to resuspend.
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.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[of resuspended NEBNext Sample Purification Beads]
Be careful to expel all of the liquid out of the tip during the last mix.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
Incubate sample on bench top for at least  at .
0 Room temperature
?. [Clean-Up of Deaminated DNA (Section 2.9)]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
After  , 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.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
Add  to the tubes while in the magnetic stand. Incubate at  for  , then carefully remove and discard the supernatant.
[of 80% freshly prepared ethanol]
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
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.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
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 brown and start to crack they are too dry.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding .
[of Elution Buffer (white)]
?. [Clean-Up of Deaminated DNA (Section 2.9)]
Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Clean-Up of Deaminated DNA (Section 2.9)]
Place the tube on the magnetic stand. After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at  .
?. [PCR Amplification (Section 2.10)]
, add the following components to the  from the previous section:  AB1COMPONENTVOLUME2EM-seq Index Primer*, **5 µl3(blue) NEBNext Q5U Master Mix25 µl4Total Volume50 µl
on ice
[of deaminated DNA]
AB1COMPONENTVOLUME2EM-seq Index Primer*, **5 µl3(blue) NEBNext Q5U Master Mix25 µl4Total Volume50 µl
?. [PCR Amplification (Section 2.10)]
Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
?. [PCR Amplification (Section 2.10)]
Place the tube in a thermocycler and perform PCR amplification using the following cycling conditions:  ABCD1CYCLE STEPTEMPTIMECYCLES2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds4-8*4Annealing62°C30 seconds5Extension65°C60 seconds6Final Extension65°C5 minutes17Hold4°C∞-
ABCD1CYCLE STEPTEMPTIMECYCLES2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds4-8*4Annealing62°C30 seconds5Extension65°C60 seconds6Final Extension65°C5 minutes17Hold4°C∞-
SAFE STOPPING POINT: Samples can be stored overnight at either   in the thermocycler or at   in the freezer.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Vortex Sample Purification Beads to resuspend.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[of water]
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Incubate samples on bench top for at least  at .
0 Room temperature
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
After , 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.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Add  to the tubes while in the magnetic stand. Incubate at  for  , then carefully remove and discard the supernatant.
[of 80% freshly prepared ethanol]
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
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.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Air dry the beads for up to  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 brown and start to crack they are too dry.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding  or .
[of Elution Buffer (white)]
[of TE 10 mM Tris, 0.1 mM EDTA, pH 8.0) or low TE (for long term storage)]
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Mix well by pipetting up and down 10 times. Incubate for at least  at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Place the tube on the magnetic stand. After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at  .
?. [Library Quantification (Section 2.12)]
Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries.50 ng of NA12878 genomic DNA.
A typical EM-seq library would have the following TapeStation trace.
?. [Library Quantification (Section 2.12)]
Sequence using the preferred Illumina platform. 2 x 100 base reads or 2 x 150 base reads for large insert libraries.
?. [Clean-Up of Amplified Libraries (Section 2.11)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[of resuspended NEBNext Sample Purification Beads]
Be careful to expel all of the liquid out of the tip during the last mix.
?. [Denaturation of DNA]
, add the following components to the .  AB1ComponentVolume2Nuclease-free water68 µl3(orange) APOBEC Reaction Buffer10 µl4(orange) BSA1 µl5(orange) APOBEC1 µl6Total Volume100 µl
on ice
[of denatured DNA]
AB1ComponentVolume2Nuclease-free water68 µl3(orange) APOBEC Reaction Buffer10 µl4(orange) BSA1 µl5(orange) APOBEC1 µl6Total Volume100 µl
For multiple reactions, a master mix of the reaction components can be prepared before addition to the denatured DNA.
?. [Denaturation of DNA]
Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
?. [Denaturation of DNA]
Incubate at  for , then at  in a thermocycler with the heated lid set to ≥ or on.
37 °C
4 °C
45 °C
SAFE STOPPING POINT: Samples can be stored overnight at either 4°C in the thermocycler or at -20°C in the freezer.
?. [Clean-Up of Deaminated DNA ]
Vortex Sample Purification Beads to resuspend.
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.
?. [Clean-Up of Deaminated DNA ]
Add  to each sample.
[of resuspended NEBNext Sample Purification Beads]
?. [Clean-Up of Deaminated DNA ]
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.
?. [Clean-Up of Deaminated DNA ]
Incubate sample on bench top for at least  at .
0 Room temperature
?. [Clean-Up of Deaminated DNA ]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Clean-Up of Deaminated DNA ]
After  ,or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets.
DO NOT discard the beads.
?. [Clean-Up of Deaminated DNA ]
Add  to the tubes while in the magnetic stand.
[of 80% freshly prepared ethanol]
?. [Clean-Up of Deaminated DNA ]
Incubate at  for  , then carefully remove and discard the supernatant.
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Clean-Up of Deaminated DNA ]
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.
?. [Clean-Up of Deaminated DNA ]
Air dry the beads for up to  while the tubes are on the magnetic stand with the lid open.
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 brown and start to crack they are too dry.
?. [Clean-Up of Deaminated DNA ]
Remove the tubes from the magnetic stand.
?. [Clean-Up of Deaminated DNA ]
Elute the DNA target from the beads by adding .
[of Elution Buffer (white)]
?. [Clean-Up of Deaminated DNA ]
Mix well by pipetting up and down 10 times.
?. [Clean-Up of Deaminated DNA ]
Incubate for at least  at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Clean-Up of Deaminated DNA ]
Place the tube on the magnetic stand.
?. [Clean-Up of Deaminated DNA ]
After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at -20°C.
?. [PCR Amplification]
, add the following components to the  from the previous section:  AB1ComponentVolume2EM-seq Index Primer*, **5 µl3(blue) NEBNext Q5U Master Mix25 µl4Total Volume50 µl
on ice
[of denatured DNA]
AB1ComponentVolume2EM-seq Index Primer*, **5 µl3(blue) NEBNext Q5U Master Mix25 µl4Total Volume50 µl
?. [PCR Amplification]
Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
?. [PCR Amplification]
Place the tube in a thermocycler and perform PCR amplification using the following cycling conditions:  ABCD1Cycle StepTempTimeCycles2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds4-8*4Annealing62°C30 seconds-5Extension65°C60 seconds-6Final Extension65°C5 minutes17Hold4°C∞-
ABCD1Cycle StepTempTimeCycles2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds4-8*4Annealing62°C30 seconds-5Extension65°C60 seconds-6Final Extension65°C5 minutes17Hold4°C∞-
SAFE STOPPING POINT: Samples can be stored overnight at either 4°Cin the thermocycler or at -20°C in the freezer.
?. [Clean-Up of Amplified Libraries]
Vortex Sample Purification Beads to resuspend.
?. [Clean-Up of Amplified Libraries]
Add  to each sample.
[resuspended NEBNext Sample Purification Beads]
?. [Clean-Up of Amplified Libraries]
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.
?. [Clean-Up of Amplified Libraries]
Add  to each sample.
[of resuspended NEBNext Sample Purification Beads]
?. [Clean-Up of Amplified Libraries]
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.
?. [Clean-Up of Amplified Libraries]
Incubate samples on bench top for at least  at .
0 Room temperature
?. [Clean-Up of Amplified Libraries]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Clean-Up of Amplified Libraries]
After , or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets.
DO NOT discard the beads.
?. [Clean-Up of Amplified Libraries]
Add  to the tubes while in the magnetic stand.
[of 80% freshly prepared ethanol]
?. [Clean-Up of Amplified Libraries]
Incubate at  for  , then carefully remove and discard the supernatant.
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Clean-Up of Amplified Libraries]
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.
?. [Clean-Up of Amplified Libraries]
Air dry the beads for up to  while the tubes are on the magnetic stand with the lid open.
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 brown and start to crack they are too dry.
?. [Clean-Up of Amplified Libraries]
Remove the tubes from the magnetic stand.
?. [Clean-Up of Amplified Libraries]
Elute the DNA target from the beads by adding  or .
[of Elution Buffer (white)]
[of TE 10 mM Tris, 0.1 mM EDTA, pH 8.0) or low TE (for long term storage)]
?. [Clean-Up of Amplified Libraries]
Mix well by pipetting up and down 10 times.
?. [Clean-Up of Amplified Libraries]
Incubate for at least  at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Clean-Up of Amplified Libraries]
Place the tube on the magnetic stand.
?. [Clean-Up of Amplified Libraries]
After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at -20°C.
?. [Library Quantification]
Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries.
A typical EM-seq library would have the following TapeStation trace.
?. [Library Quantification]
Sequence using the preferred Illumina platform. 2 x 76 base reads or 2 x 100 base reads for standard sized libraries.
?. [Denaturation of DNA]
, add the following components to the .  AB1ComponentVolume2Nuclease-free water68 µl3(orange) APOBEC Reaction Buffer10 µl4(orange) BSA1 µl5(orange) APOBEC1 µl6Total Volume100 µl
on ice
[of denatured DNA]
AB1ComponentVolume2Nuclease-free water68 µl3(orange) APOBEC Reaction Buffer10 µl4(orange) BSA1 µl5(orange) APOBEC1 µl6Total Volume100 µl
For multiple reactions, a master mix of the reaction components can be prepared before addition to the denatured DNA.
?. [Denaturation of DNA]
Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
?. [Denaturation of DNA]
Incubate at  for , then at  in a thermocycler with the heated lid set to ≥ or on.
37 °C
4 °C
45 °C
SAFE STOPPING POINT: Samples can be stored overnight at either 4°C in the thermocycler or at -20°C in the freezer.
?. [Clean-Up of Deaminated DNA]
Vortex Sample Purification Beads to resuspend.
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.
?. [Clean-Up of Deaminated DNA]
Add  to each sample.
[of resuspended NEBNext Sample Purification Beads]
?. [Clean-Up of Deaminated DNA]
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.
?. [Clean-Up of Deaminated DNA]
Incubate sample on bench top for at least  at .
0 Room temperature
?. [Clean-Up of Deaminated DNA]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Clean-Up of Deaminated DNA]
After  ,or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets.
DO NOT discard the beads.
?. [Clean-Up of Deaminated DNA]
Add  to the tubes while in the magnetic stand.
[of 80% freshly prepared ethanol]
?. [Clean-Up of Deaminated DNA]
Incubate at  for  , then carefully remove and discard the supernatant.
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Clean-Up of Deaminated DNA]
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.
?. [Clean-Up of Deaminated DNA]
Air dry the beads for up to  while the tubes are on the magnetic stand with the lid open.
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 brown and start to crack they are too dry.
?. [Clean-Up of Deaminated DNA]
Remove the tubes from the magnetic stand.
?. [Clean-Up of Deaminated DNA]
Elute the DNA target from the beads by adding .
[of Elution Buffer (white)]
?. [Clean-Up of Deaminated DNA]
Mix well by pipetting up and down 10 times.
?. [Clean-Up of Deaminated DNA]
Incubate for at least  at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Clean-Up of Deaminated DNA]
Place the tube on the magnetic stand.
?. [Clean-Up of Deaminated DNA]
After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at -20°C.
?. [PCR Amplification]
, add the following components to the  from the previous section:  AB1ComponentVolume2EM-seq Index Primer*, **5 µl3(blue) NEBNext Q5U Master Mix25 µl4Total Volume50 µl
on ice
[of denatured DNA]
AB1ComponentVolume2EM-seq Index Primer*, **5 µl3(blue) NEBNext Q5U Master Mix25 µl4Total Volume50 µl
?. [PCR Amplification]
Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
?. [PCR Amplification]
Place the tube in a thermocycler and perform PCR amplification using the following cycling conditions:  ABCD1Cycle StepTempTimeCycles2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds4-8*4Annealing62°C30 seconds-5Extension65°C60 seconds-6Final Extension65°C5 minutes17Hold4°C∞-
ABCD1Cycle StepTempTimeCycles2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds4-8*4Annealing62°C30 seconds-5Extension65°C60 seconds-6Final Extension65°C5 minutes17Hold4°C∞-
SAFE STOPPING POINT: Samples can be stored overnight at either 4°Cin the thermocycler or at -20°C in the freezer.
?. [Clean-Up of Amplified Libraries]
Vortex Sample Purification Beads to resuspend.
?. [Clean-Up of Amplified Libraries]
Add  to each sample.
[resuspended NEBNext Sample Purification Beads]
?. [Clean-Up of Amplified Libraries]
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.
?. [Clean-Up of Amplified Libraries]
Add  to each sample.
[of resuspended NEBNext Sample Purification Beads]
?. [Clean-Up of Amplified Libraries]
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.
?. [Clean-Up of Amplified Libraries]
Incubate samples on bench top for at least  at .
0 Room temperature
?. [Clean-Up of Amplified Libraries]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Clean-Up of Amplified Libraries]
After , or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets.
DO NOT discard the beads.
?. [Clean-Up of Amplified Libraries]
Add  to the tubes while in the magnetic stand.
[of 80% freshly prepared ethanol]
?. [Clean-Up of Amplified Libraries]
Incubate at  for  , then carefully remove and discard the supernatant.
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Clean-Up of Amplified Libraries]
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.
?. [Clean-Up of Amplified Libraries]
Air dry the beads for up to  while the tubes are on the magnetic stand with the lid open.
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 brown and start to crack they are too dry.
?. [Clean-Up of Amplified Libraries]
Remove the tubes from the magnetic stand.
?. [Clean-Up of Amplified Libraries]
Elute the DNA target from the beads by adding  or .
[of Elution Buffer (white)]
[of TE 10 mM Tris, 0.1 mM EDTA, pH 8.0) or low TE (for long term storage)]
?. [Clean-Up of Amplified Libraries]
Mix well by pipetting up and down 10 times.
?. [Clean-Up of Amplified Libraries]
Incubate for at least  at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Clean-Up of Amplified Libraries]
Place the tube on the magnetic stand.
?. [Clean-Up of Amplified Libraries]
After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at -20°C.
?. [Library Quantification]
Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries.
A typical EM-seq library would have the following TapeStation trace.
?. [Library Quantification]
Sequence using the preferred Illumina platform. 2 x 76 base reads or 2 x 100 base reads for standard sized libraries.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
, add the following components directly to the .  AB1ComponentsVolume2(yellow) TET2 Reaction Buffer (TET2 Reaction Buffer plus reconstituted TET2 Reaction Buffer Supplement)10 µl3(yellow) Oxidation Supplement1 µl4(yellow) DTT1 µl5(yellow) Oxidation Enhancer1  µl6(yellow) TET24 µlMix thoroughly by vortexing, centrifuge briefly.
on ice
[EM-seq adaptor ligated]
AB1ComponentsVolume2(yellow) TET2 Reaction Buffer (TET2 Reaction Buffer plus reconstituted TET2 Reaction Buffer Supplement)10 µl3(yellow) Oxidation Supplement1 µl4(yellow) DTT1 µl5(yellow) Oxidation Enhancer1  µl6(yellow) TET24 µl
For multiple reactions, a master mix of the reaction components can be prepared before addition to the sample DNA. 5mC/5hmC oxidation is initiated by the addition of the Fe(II) solution to the reaction after the addition of master mix.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Dilute the  by adding  to .Combine Diluted Fe(II) Solution and EM-seq DNA with Oxidation Enzymes (from Step 25).  AB1ComponentVolume2 EM-seq DNA 45 μl3Diluted Fe(II) Solution 5 μl4Total Volume50 μlMix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
[Fe(II) Solution (yellow)]
1 µl
[of water]
Use the solution immediately, do not store it. Discard after use.
AB1ComponentVolume2 EM-seq DNA 45 μl3Diluted Fe(II) Solution 5 μl4Total Volume50 μl
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Incubate at  for  in a thermocycler with the heated lid set to ≥   or on.
37 °C
45 °C
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Transfer the samples to ice and add  (yellow).  AB1ComponetVolume2(yellow) Stop Reagent1 μl3Total Volume51 μlMix thoroughly by vortexing or by pipetting up and down at least 10 times and centrifuge briefly.
[of Stop Reagent]
AB1ComponetVolume2(yellow) Stop Reagent1 μl3Total Volume51 μl
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Incubate at  for   then at  in the thermocycler with the heated lid set to ≥  or on.
37 °C
4 °C
45 °C
SAFE STOPPING POINT: Samples can be stored overnight at either   in the thermocycler or at   in the freezer.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Vortex Sample Purification Beads to resuspend.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[of resuspended NEBNext Sample Purification Beads]
Be careful to expel all of the liquid out of the tip during the last mix.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Incubate samples on bench top for at least 5 minutes at .
0 Room temperature
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
After , or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets
Do not discard the beads.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Add  to the tubes while in the magnetic stand. Incubate at  for  , then carefully remove and discard the supernatant.
[of 80% freshly prepared ethanol]
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
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.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
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 brown and start to crack they are too dry.
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 brown and start to crack they are too dry.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding .
[of Elution Buffer (white)]
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Place the tube on the magnetic stand. After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at  .
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Use option A for denaturing using Formamide and option B for denaturing using 0.1 N Sodium hydroxide.
The DNA can be denatured using either Formamide or 0.1 N Sodium Hydroxide.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
, add the following components to the .  AB1ComponentVolume2Nuclease-free water68 µl3(orange) APOBEC Reaction Buffer10 µl4(orange) BSA1 µl5(orange) APOBEC1 µl6Total Volume100 µl
on ice
[of denatured DNA]
AB1ComponentVolume2Nuclease-free water68 µl3(orange) APOBEC Reaction Buffer10 µl4(orange) BSA1 µl5(orange) APOBEC1 µl6Total Volume100 µl
For multiple reactions, a master mix of the reaction components can be prepared before addition to the denatured DNA.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Incubate at  for , then at  in a thermocycler with the heated lid set to ≥ or on.
37 °C
4 °C
45 °C
SAFE STOPPING POINT: Samples can be stored overnight at either   in the thermocycler or at   in the freezer.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Vortex Sample Purification Beads to resuspend.
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.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[of resuspended NEBNext Sample Purification Beads]
Be careful to expel all of the liquid out of the tip during the last mix.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Incubate sample on bench top for at least 5 minutes at .
0 Room temperature
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
After  ,or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets.
DO NOT discard the beads.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Add  to the tubes while in the magnetic stand. Incubate at  for  , then carefully remove and discard the supernatant.
[of 80% freshly prepared ethanol]
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
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.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Air dry the beads for up to 90 minutes while the tubes are on the magnetic stand with the lid open.
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 brown and start to crack they are too dry.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding .
[of Elution Buffer (white)]
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Mix well by pipetting up and down 10 times. Incubate for at least 1 minute at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Place the tube on the magnetic stand. After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at  .
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
, add the following components to the  from the previous section:  AB1ComponentVolume2EM-seq Index Primer*, **5 µl3(blue) NEBNext Q5U Master Mix25 µl4Total Volume50 µl
on ice
[of deaminated DNA]
AB1ComponentVolume2EM-seq Index Primer*, **5 µl3(blue) NEBNext Q5U Master Mix25 µl4Total Volume50 µl
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Place the tube in a thermocycler and perform PCR amplification using the following cycling conditions:  ABCD1Cycle StepTempTimeCycles2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds4-8*4Annealing62°C30 seconds4-8*5Extension65°C60 seconds4-8*6Final Extension65°C5 minutes17Hold4°C∞-
ABCD1Cycle StepTempTimeCycles2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds4-8*4Annealing62°C30 seconds4-8*5Extension65°C60 seconds4-8*6Final Extension65°C5 minutes17Hold4°C∞-
SAFE STOPPING POINT: Samples can be stored overnight at either   in the thermocycler or at   in the freezer.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Vortex Sample Purification Beads to resuspend.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[resuspended NEBNext Sample Purification Beads]
Be careful to expel all of the liquid out of the tip during the last mix.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Add  to each sample. Mix well by pipetting up and down at least 10 times.
[of resuspended NEBNext Sample Purification Beads]
Be careful to expel all of the liquid out of the tip during the last mix.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Incubate samples on bench top for at least 5 minutes at .
0 Room temperature
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
After , or when the solution is clear, carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets.
DO NOT discard the beads.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Add  to the tubes while in the magnetic stand. Incubate at  for  , then carefully remove and discard the supernatant.
[of 80% freshly prepared ethanol]
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
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.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Air dry the beads for up to 2 minutes while the tubes are on the magnetic stand with the lid open.
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 brown and start to crack they are too dry.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding  or .
[of Elution Buffer (white)]
[of TE 10 mM Tris, 0.1 mM EDTA, pH 8.0) or low TE (for long term storage)]
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Mix well by pipetting up and down 10 times. Incubate for at least  at .
0 Room temperature
If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing back on the magnetic stand.
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Place the tube on the magnetic stand. After , or whenever the solution is clear, transfer  to a new PCR tube.
[of the supernatant]
SAFE STOPPING POINT: Samples can be stored overnight at  .
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Use a Bioanalyzer or TapeStation to determine the size distribution and concentration of the libraries.
A typical EM-seq library would have the following TapeStation trace.
Refer to the graph located in the original protocol found here: https://www.neb.com/protocols/2019/03/28/protocol-for-use-with-standard-insert-libraries-370-420-bp-e7120
?. [Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines (Section 1.5)]
Sequence using the preferred Illumina platform. 2 x 76 base reads or 2 x 100 base reads for standard sized libraries.
?. [Clean-Up of Adaptor Ligated DNA (Section 2.4)]
Incubate at  for  before carefully removing and discarding the supernatant.
0 Room temperature
Be careful not to disturb the beads that contain DNA targets.