TITLE: Protocol for Use with Standard Insert Libraries (370-420 bp) (NEB#E7120)
AUTHORS: New England   Biolabs

[DESCRIPTION]
This protocol details how to construct DNA libraries from start to finish using NEBNext reagents.

The corresponding NEB manual is here: https://www.neb.com/-/media/nebus/files/manuals/manuale7120.pdf and this protocol relates to section 1.

Overview

 
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). 

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.

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 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.

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) -> 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.

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 with Standard Size Libraries (370–420 bp). If following the Section 2 Protocol, for use with Large Size Libraries (470–520 bp), users need to supply 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 with the 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.

[BEFORE_START]
Safe Stopping Point: 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.

Caution: This signifies a step in the protocol that has two paths leading to the same end point.

(Color): Colors in parenthesis indicate the cap color of the reagent to be added.

Starting Material: 10 ng–200 ng DNA

[STEPS]
SECTION: DNA Preparation
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).
 
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 with read 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.
 
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 by deeper 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.
SECTION: End Prep of Sheared DNA
3. on ice, mix the following components in a sterile nuclease-free PCR tube:
 
Note: NEBNext Ultra II End Prep Reaction Buffer and Enzyme Mix can be pre-mixed ahead of time as a master mix.

SECTION: Ligation of EM-seq Adaptor
6. on ice, add the following components directly to the End Prep reaction mixture and mix well:

 

Note: Ligation Enhancer and Ligation 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 the other ligation reagents.

SECTION: Ligation of EM-seq Adaptor
7. Set a 100 µL or 200 µL pipette to 80 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.

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.


SECTION: Ligation of EM-seq Adaptor
8. 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.
SECTION: Clean-Up of Adaptor Ligated DNA
9. Vortex Sample Purification Beads to resuspend.
SECTION: Clean-Up of Adaptor Ligated DNA
10. Add 110 µl (~1.1X) 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.

SECTION: Clean-Up of Adaptor Ligated DNA
11. Incubate samples on bench top for at least 5 min at Room temperature.

SECTION: Clean-Up of Adaptor Ligated DNA
12. Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
SECTION: Clean-Up of Adaptor Ligated DNA
13. After 5 min (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.


SECTION: Clean-Up of Adaptor Ligated DNA
14. Add 200 µL  to the tubes while in the magnetic stand. Incubate at Room temperature for 30 s and then carefully removing and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Clean-Up of Adaptor Ligated DNA
15. 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.

SECTION: Clean-Up of Adaptor Ligated DNA
16. 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 brown and start to crack they are too dry.

 


SECTION: Clean-Up of Adaptor Ligated DNA
17. Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 29 µL (white).
SECTION: Clean-Up of Adaptor Ligated DNA
18. Mix well by pipetting up and down 10 times. Incubate for at least 1 min at 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.


SECTION: Clean-Up of Adaptor Ligated DNA
19.  Place the tube on the magnetic stand. After 3 min, or whenever the solution is clear, transfer 28 µL to a new PCR tube.
 

SECTION: Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
20. Prepare TET2 Buffer. Use option A if you have E7120S/ E7120G (24 Reactions/G size) and option B if you have E7120L (96 reactions).
 




SECTION: Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
23. on ice, add the following components directly to the EM-seq adaptor ligated DNA. 

 

Mix thoroughly by vortexing, centrifuge briefly. For multiple reactions, a master mix of the above 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.

SECTION: Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
24. Dilute the 500 mM (yellow) Fe(II) Solution by adding 1 µL to 1249 µL.
 

Combine Diluted (yellow) Fe(II) Solution and Reaction DNA Oxidation Enzymes as described below. 

 

Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
SECTION: Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
25. 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
SECTION: Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
26. Transfer the samples to ice and add 1 µL (yellow).
 

Mix thoroughly by vortexing or by pipetting up and down at least 10 times and centrifuge briefly.
SECTION: Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
27. 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

 

SECTION: Clean-Up of TET2 Converted DNA
28. Vortex Sample Purification Beads to resuspend.
SECTION: Clean-Up of TET2 Converted DNA
29. Add 90 µl (1.8X) 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.

SECTION: Clean-Up of TET2 Converted DNA
30. Incubate samples on bench top for at least 5 min at Room temperature.

SECTION: Clean-Up of TET2 Converted DNA
31. Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
SECTION: Clean-Up of TET2 Converted DNA
32. After 5 min (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.
SECTION: Clean-Up of TET2 Converted DNA
33. Add 200 µL to the tubes while in the magnetic stand. Incubate at Room temperature for 30 s , then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Clean-Up of TET2 Converted DNA
34. 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. 

SECTION: Clean-Up of TET2 Converted DNA
35. 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 brown and start to crack they are too dry.
 


SECTION: Clean-Up of TET2 Converted DNA
36. Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 17 µL.
SECTION: Clean-Up of TET2 Converted DNA
37. Mix well by pipetting up and down 10 times. Incubate for at least 1 min at 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.

SECTION: Clean-Up of TET2 Converted DNA
38. Place the tube on the magnetic stand. After 3 min (or whenever the solution is clear), transfer 16 µL to a new PCR tube.
 

SECTION: Denaturation of DNA
39. 
 

Use Option A for denaturing using Formamide and Option B for denaturing using 0.1 N Sodium hydroxide.
SECTION: End Prep of Sheared DNA
5. Place in a thermal cycler with the heated lid set to ≥ 75 °C or on, and run the following program:

30 min at 20 °C 
30 min at 65 °C 
Hold at 4 °C 
SECTION: End Prep of Sheared DNA
4. 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.

SECTION: Shearing DNA
2. 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
SECTION: Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
21. Option A: E7120S/E7120G
Add 100 µL to one tube of TET2 Reaction Buffer Supplement and mix well. Write date on tube.
SECTION: Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines
22. Option B: E7120L
Add 400 µL to one tube of TET2 Reaction Buffer Supplement and mix well. Write date on tube.

The reconstituted buffer should be stored at -20 °C and discarded after 4 months.
SECTION: Library Quantification
77. 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.

 

Sequence using the preferred Illumina platform. 2 x 76 base reads or 2 x 100 base reads for standard sized libraries.
SECTION: Clean-Up of Amplified Libraries
76. Place the tube on the magnetic stand. After 3 min (or whenever the solution is clear), transfer 20 µL to a new PCR tube.
 

SECTION: Clean-Up of Amplified Libraries
75. Mix well by pipetting up and down 10 times. Incubate for at least 1 min at 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.
SECTION: Clean-Up of Amplified Libraries
74. Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 21 µL For long terms storage use,  21 µl of 1XTE (10 mM Tris, 1 mM EDTA, pH 8.0), Low TE (10 mM Tris, 0.2 mM EDTA, pH 8.0) or 0.1XTE (1 mM Tris, 0.1 mM EDTA, pH 8.0).
SECTION: Clean-Up of Amplified Libraries
73. Air dry the beads for up to 2 min 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.
 



SECTION: Clean-Up of Amplified Libraries
72. 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.

SECTION: Clean-Up of Amplified Libraries
71. Add 200 µL to the tubes while in the magnetic stand. Incubate at Room temperature for 30 s , then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
SECTION: Clean-Up of Amplified Libraries
70. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

 



SECTION: Clean-Up of Amplified Libraries
69. Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
SECTION: Clean-Up of Amplified Libraries
68. Incubate samples on bench top for at least 5 min at Room temperature.
SECTION: Clean-Up of Amplified Libraries
67. Add 45 µl (0.9X) 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.
SECTION: Clean-Up of Amplified Libraries
66. Vortex Sample Purification Beads to resuspend.
SECTION: PCR Amplification
65. DenPlace the tube in a thermal cycler with the heated lid set to 105°C and perform PCR amplification using the following cycling conditions:

 
*Cycle Recommendations:
10 ng DNA input: 8 cycles
50 ng DNA input: 5-6 cycles
200 ng DNA input: 4 cycles

Safe Stopping Point: Samples can be stored overnight at either 4°C in the thermal cycler or at -20°C in the freezer. 

SECTION: PCR Amplification
64. Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
SECTION: Clean up of Deaminated DNA
62. Place the tube on the magnetic stand. After 3 min (or whenever the solution is clear), transfer 20 µL to a new PCR tube.
 

SECTION: Clean up of Deaminated DNA
61. Mix well by pipetting up and down 10 times. Incubate for at least 1 min at 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.

SECTION: Clean up of Deaminated DNA
60. Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 21 µL.
SECTION: Clean up of Deaminated DNA
59. Air dry the beads for up to 90 s 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.
 



SECTION: Clean up of Deaminated DNA
58. 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.


SECTION: Clean up of Deaminated DNA
57. Add 200 µL to the tubes while in the magnetic stand. Incubate at Room temperature for 30 s , then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Clean up of Deaminated DNA
56. After 5 min  (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. 
 


SECTION: Clean up of Deaminated DNA
55. Place the tubes against an appropriate magnetic stand to separate the beads from the supernatant.
SECTION: Clean up of Deaminated DNA
54. Incubate sample on bench top for at least 5 min at Room temperature.

SECTION: Clean up of Deaminated DNA
53. Add 100 µl (1.0X) 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.
SECTION: Clean up of Deaminated DNA
52. 
 

Vortex Sample Purification Beads to resuspend.
SECTION: Deamination of Cytosines
51. Place in a thermal cycler, and run the following program wiht the heated lid set to ≥ 45 °Cor on.
180 minat 37 °C
Hold at 4 °C 

 
 
SECTION: Deamination of Cytosines
50. Mix thoroughly by vortexing or by pipetting up and down at least 10 times, centrifuge briefly.
SECTION: Deamination of Cytosines
49. on ice, add the following components to the denatured DNA.

 

 

SECTION: Option A: Formamide (Recommended)
40. Pre-heat thermal cycler to 85 °C with the heated lid set to ≥ 105°C or on.
SECTION: Option A: Formamide (Recommended)
41. Add 4 µL to the 16 µL(Step 39). Vortex to mix or by pipetting up and down at least 10 times, centrifuge briefly.
SECTION: Option A: Formamide (Recommended)
42. Incubate at 85 °C for 10 min in the pre-heated thermal cycler.
SECTION: Option A: Formamide (Recommended)
43. Immediately place on ice and allow the sample to fully cool (~2 minutes) before proceeding to Deamination of Cytosines.
SECTION: Option B: Sodium Hydroxide (Optional, see FAQ at NEB.com about preparing NaOH)
44. Prepare freshly diluted 0.1 N NaOH.
SECTION: Option B: Sodium Hydroxide (Optional, see FAQ at NEB.com about preparing NaOH)
45. Pre-heat thermal cycler to 50 °C with the heated lid set to ≥  105 °C or on.
SECTION: Option B: Sodium Hydroxide (Optional, see FAQ at NEB.com about preparing NaOH)
46. Add 4 µL to the 16 µL (Step 39). Vortex to mix or by pipetting up and down at least 10 times, centrifuge briefly.
SECTION: Option B: Sodium Hydroxide (Optional, see FAQ at NEB.com about preparing NaOH)
47. Incubate at 50 °C for 10 min in the pre-heated thermal cycler.
SECTION: Option B: Sodium Hydroxide (Optional, see FAQ at NEB.com about preparing NaOH)
48. Immediately place on ice and allow the sample to fully cool (~2 minutes) before proceeding to Deamination of Cytosines.
SECTION: PCR Amplification
63. On ice, add the following components to the deaminated DNA:
 
*Refer to Section 3 in the manual at NEB.com 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.