Protocol for use with Large Insert Libraries (470–520 bp)  (NEB #E7120)

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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. 
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Starting Material: 10 ng–200 ng DNA

2.1 DNA Preparation

2.1.1. DNA and Control DNAs

Combine genomic DNA (10–200 ng) with control DNAs, CpG methylated pUC19  (lilac) and unmethylated lambda DNA (lilac) in 50 µl made up with 0.1X TE pH 8.0. The amount of control DNA added is dependent on the number of reads required.

If checking library quality in 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 deepsequencing. Having higher ng of control DNA for samples that are sequenced on a MiSeq ensures that there are enough controlreads to accurately call cytosine conversion. We recommend this for users who are inexperienced with next generationsequencing library preparation. For libraries sequenced to a depth of 2–4 M paired end reads, approximately 5,000 x 76 basepaired 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:

  • 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  control DNA(0.001 ng) and 1 µl diluted unmethylated lambda DNA (0.02 ng) per 10–200 ng sample DNA.

2.1.2. Shearing DNA

The combined 50 µl genomic DNA 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.

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:

COMPONENT

VOLUME

Fragmented DNA

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

2.2.2 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.

2.2.3. Place in a thermocycler, and run the following program:

30 minutes @ 20°C

30 minutes @ 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 60 µl End Prep reaction mixture and mix well:

COMPONENT

VOLUME

 (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 µl

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.

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 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.

2.3.3. Incubate at 20°C for 15 minutes in a thermocycler.

 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 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.

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

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 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.

2.4.9. Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 30 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 the sample 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 29 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 have E7120S (24 Reactions) and option B if you have E7120L (96 reactions).

Note: The TET2 Reaction Buffer Supplement is a powder. Centrifuge before use to ensure it is at the bottom of the tube.

2.5.1A. Add 100 µl of TET2 Reaction Buffer to one tube of TET2 Reaction Buffer Supplement and mix well.

2.5.1B. Add 400 µl of TET2 Reaction Buffer to one tube of TET2 Reaction Buffer Supplement and mix well.

NOTE: The reconstituted buffer should be stored at -20°C and discarded after 4 months.

2.5.2. Dilute the 500 mM Fe(II) Solution  (yellow) by adding 1 µl to 1249 µl of water.

NOTE: Use the solution immediately, do not store it. Discard after use.

2.5.3. On ice, add the following components directly to the 29 µl EM-seq adaptor ligated DNA (from Step 2.4.11).

COMPONENT

VOLUME

 (yellow) TET2 Reaction Buffer (reconstituted)

10 µl

 (yellow) Oxidation Supplement

1 µl

 (yellow) Oxidation Enhancer

1 µl

 (yellow) TET2

4 µl

Mix thoroughly by vortexing, centrifuge briefly, then add

COMPONENT

VOLUME

Diluted Fe(II) Solution (from Step 2.5.2)

5 µl

Total Volume

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

2.5.4. Incubate at 37°C for 1 hour in a thermocycler.

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

COMPONENT

VOLUME

 (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. Incubate at 37°C for 30 minutes then at 4°C in a thermocycler.

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

2.6 Clean-Up of TET2 Converted DNA

2.6.1. Vortex Sample Purification Beads to resuspend.

2.6.2. Add 90 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.

2.6.3. 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 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.

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

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 the sample 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.

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

2.7A.1. Pre-heat thermocycler to 85°C.

2.7A.2. Add 4 µl Formamide to the 16 µl of oxidized DNA. 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 thermocycler.

2.7A.4. Immediately place on ice.

2.7A.5. Proceed immediately to Section 2.8.

2.7B: Sodium Hydroxide

2.7B.1. Prepare freshly diluted 0.1 N NaOH.

2.7B.2. Pre-heat thermocycler to 50°C.

2.7B.3. Add 4 µl 0.1 N NaOH to the 16 µl of oxidized DNA. 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 thermocycler.

2.7B.5. Immediately place on ice.

2.7B.6. Proceed immediately to Section 2.8.

2.8 Deamination of Cytosines

2.8.1. On ice, add the following components to the 20 µl of denatured DNA.

COMPONENT

VOLUME

Nuclease-free water

68 µl

 (orange) APOBEC Reaction Buffer

10 µl

 (orange)BSA

1 µl

 (orange)APOBEC

1 µl

Total volume

100 µl

For 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. Incubate at 37°C for 3 hours then at 4°C in a thermocycler.

 Safe Stopping Point: Samples can be stored overnight at either 4°C in the thermocycler 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 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.

2.9.3. 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.

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 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.

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

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 the sample 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 20 µl of deaminated DNA from Step 2.9.11.

COMPONENT

VOLUME

EM-seq Index Primer*,**

5 µl

 (blue) NEBNext Q5U Master Mix

25 µl

Total volume

50 µl

* Refer to manual for barcode pooling guidelines.

** EM-seq primers are supplied in tubes in #E7120S or as a 96 Unique Dual Index Primer Pairs Plate in #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 thermocycler 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

4-8*

Annealing

62°C

30 seconds

Extension

65°C

60 seconds

Final Extension

65°C

5 minutes

1

Hold

4°C

*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 thermocycler 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.

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 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 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.

2.11.10. Remove the tubes from the magnetic stand. Elute the DNA target from the beads by adding 21 μl of Elution Buffer  (white) or 10 mM Tris, 0.1 mM EDTA, pH 8.0 (for long term storage). 

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 the sample 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.

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 library would 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 standard sized libraries.