Protocol for EM-seq Conversion Module (NEB #E7125)

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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 fragmented double stranded DNA

1. DNA Preparation

1.1. DNA and Control DNA

DNA should not contain any EDTA moving into the Oxidation Reaction and should be in 28 µl of water or 10mM Tris pH 8.0. Control DNAs for assessing oxidation and deamination are included with this module and their use is dependent on the users requirements. For sequencing on an Illumina platform, refer to the Enzymatic Methyl-seq Kit Manual (NEB #E7120) for usage recommendations. For other downstream applications and sequencing platforms, please refer to manufacturer's guidelines.

2 Oxidation of 5-Methylcytosines and 5-Hydroxymethylcytosines

2.1. Prepare TET2 Buffer. Use option A if you have E7125S/E7125G (24 Reactions/G Size) and option B if you have E7125L (96 reactions).

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

2.1A. Add 100 µl of TET2 Reaction Buffer to one tube of TET2 Reaction Buffer Supplement and mix well. Write date on tube.

2.1B. Add 400 µl of TET2 Reaction Buffer to one tube of TET2 Reaction Buffer Supplement and mix well. Write date on tube.

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

COMPONENT	VOLUME
(yellow) TET2 Reaction Buffer (TET2 Reaction Buffer plus reconstituted TET2 Reaction Buffer Supplement)	10 µl
(yellow) Oxidation Supplement	1 µl
(yellow) DTT	1 μl
(yellow) Oxidation Enhancer	1 µl
(yellow) TET2	4 µl

Mix thoroughly by vortexing, centrifuge briefly. 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.

2.3. 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.

Combine Diluted Fe(II) Solution and EM-seq DNA with Oxidation Enzymes (from Step 2.2.).

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

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

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

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

3. Clean-Up of TET2 Converted DNA

3.1. Vortex Sample Purification Beads to resuspend. SPRIselect or AMPure XP Beads can be used as well. If using AMPure XP beads, allow the beads to warm to room temperature for at least 30 minutes before use.

3.2. 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.

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

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

3.5. 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).

3.6. 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.

3.7. 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.

3.8. Air dry the beads for up to 2 minutes while the tubes are on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

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

3.10. 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.

3.11. 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.

 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.

4A: Formamide (Recommended)

4A.1. Pre-heat thermocycler to 85°C with the heated lid on.

4A.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.

4A.3. Incubate at 85°C for 10 minutes in the pre-heated thermocycler with the heated lid on.

4A.4. Immediately place on ice.

4A.5. Proceed immediately to Section 5.

4B: Sodium Hydroxide (Optional, See FAQ abaout preparing NaOH)

4B.1. Prepare freshly diluted 0.1 N NaOH.

4B.2. Pre-heat thermocycler to 50°C with the heated lid set to ≥  60°C or on.

4B.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.

4B.4. Incubate at 50°C for 10 minutes in the pre-heated thermocycler.

4B.5. Immediately place on ice.

4B.6. Proceed immediately to Section 5.

5. Deamination of Cytosines

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

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

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

5.3. Incubate at 37°C for 3 hours then at 4°C in a thermocycler with the heated lid set to ≥  45°C or on.

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

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.

6.1. Vortex Sample Purification Beads to resuspend. SPRIselect or AMPure XP Beads can be used as well. If using AMPure XP beads, allow the beads to warm to room temperature for at least 30 minutes before use.

6.2. 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.

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

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

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).

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.

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.

6.8  Air dry the beads for up to 90 seconds while the tubes are on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

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

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.

6.11. 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.

Note: Please see NEB #E7120 for the PCR amplification protocol for downstream sequencing on an Illumina platform.

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