Protocol for use with NEBNext FFPE DNA Repair Mix (M6630) and NEBNext DNA Library Prep Master Mix Set for Illumina (E6040)

Symbols
This caution sign signifies a step in the protocol that has multiple paths
leading to the same end point but is dependent on a user variable, like the
amount of input DNA.

Colored bullets indicate the cap color of the reagent to be added to a reaction.

Starting Material: 50 ng–1 μg of fragmented FFPE DNA.

3.1. NEBNext FFPE Repair

3.1.1. Mix the following components in a sterile nuclease-free tube:

FFPE DNA 53.5 µl
 (green) FFPE DNA Repair Buffer (10X) 6.5 µl
 (green) NEBNext FFPE DNA Repair Mix 2 µl
Total Volume 62 µl

3.1.2. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.

3.1.3. Incubate at 20°C for 15 minutes.

 

3.2. End Repair of Fragmented DNA

3.2.1. Add the following components directly to the FFPE repair reaction mixture and mix well:

 (green) NEBNext End Repair Reaction Buffer (10X) 3.5 µl
 (green) NEBNext End Repair Enzyme Mix 5 µl
Sterile H20 29.5 µl
Repaired DNA from above (Step 3.1.3) 62 µl
Total Volume 100 µl

3.2.2. Incubate in a thermocycler for 30 minutes at 20°C.

 

3.3. Cleanup Using AMPure XP Beads

3.3.1. Vortex AMPure XP Beads to resuspend.

3.3.2. Add 160 μl (1.6X) of resuspended AMPure XP Beads to the End Repair reaction. Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times.

3.3.3. Incubate for 5 minutes at room temperature.

3.3.4. Put the tube/PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.

3.3.5. Add 200 μl of 80% freshly prepared ethanol to the tube/PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

3.3.6. Repeat Step 3.3.5 once.

3.3.7. Air dry beads for up to 5 minutes while the tube/PCR plate is 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.

3.3.8. Remove the tube/plate from the magnet. Elute DNA target by adding 45 μl 0.1X TE or 10 mM Tris-HCl pH 8.0 to the beads. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature. Put the tube/PCR plate in the magnetic stand until the solution is clear.

3.3.9. Without disturbing the bead pellet, carefully transfer 42 μl of the supernatant to a fresh, sterile microfuge tube.

 

3.4. dA-Tailing of End Repaired DNA

3.4.1. Mix the following components in a sterile microfuge tube:

End Repaired, Blunt DNA  42 μl
 (yellow) NEBNext dA-Tailing Reaction Buffer (10X)
5 μl
 (yellow) Klenow Fragment (3´→ 5´ exo
3 μl
Total Volume 50 µl

3.4.2. Incubate in a thermocycler for 30 minutes at 37°C.

 

3.5. Cleanup Using AMPure XP Beads

3.5.1. Vortex AMPure XP Beads to resuspend.

3.5.2. Add 90 μl (1.8X) of resuspended AMPure XP Beads to the dA-Tailing reaction. Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times.

3.5.3. Incubate for 5 minutes at room temperature.

3.5.4. Put the tube/PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.

3.5.5. Add 200 μl of 80% freshly prepared ethanol to the tube/PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

3.5.6. Repeat Step 3.5.5 once.

3.5.7. Air dry beads for up to 5 minutes while the tube/PCR plate is 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.

3.5.8. Remove the tube/plate from the magnet. Elute DNA target by adding 37 μl 0.1X TE or 10 mM Tris-HCl pH 8.0 to the beads. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature. Put the tube/PCR plate in the magnetic stand until the solution is clear.

3.5.9. Without disturbing the bead pellet, carefully transfer 32.5 μl of the supernatant to a fresh, sterile microfuge tube.

 

3.6. Adaptor Ligation of dA-Tailed DNA

 If DNA input is < 100 ng, dilute the NEBNext Adaptor for Illumina (provided at 15 μM) 10 fold in 10 mM Tris-HCl pH 7.5 with 10 mM NaCl to a final concentration of 1.5 μM; use immediately.

3.6.1. Mix the following components in a sterile microfuge tube:

dA-Tailed DNA 32.5 μl  
 (red) Quick Ligation Reaction Buffer (5X) 10 μl
 (red) NEBNext Adaptor (15 μM)*   2.5 μl
 (red) Quick T4 DNA Ligase 5 μl
Total Volume 50 μl

*The NEBNext adaptor is provided in the NEBNext oligos kit. NEB has several oligo kit options, which are supplied separately from the library prep kit.

3.6.2. Incubate in a thermal cycler for 15 minutes at 20°C.

3.6.3. Add 3 μl of (red) USER Enzyme Mix by pipetting up and down, and incubate at 37°C for 15 minutes.
Note: This step is for use with NEBNext adaptors only. USER Enzyme can be found in the NEBNext Singleplex or Multiplex Oligos for Illumina.

A precipitate can form upon thawing of the NEBNext Q5 Hot Start HiFi PCR Master Mix. To ensure optimal performance, place the master mix at room temperature while performing cleanup of adaptor-ligated DNA. Once thawed, gently mix by inverting the tube several times.

 

3.7. Cleanup of Adaptor-ligated DNA

3.7.1. Vortex AMPure XP Beads to resuspend

3.7.2. Add 53 μl (1X) of resuspended AMPure XP Beads to the ligation reaction (~53 μl). Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times.

3.7.3. Incubate for 5 minutes at room temperature.

3.7.4. Put the tube/PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.

3.7.5. Add 200 μl of 80% freshly prepared ethanol to the tube/PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

3.7.6. Repeat Step 3.7.5 once.

3.7.8. Air dry beads for up to 5 minutes while the tube/PCR plate is 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.

3.7.9. Remove the tube/plate from the magnet. Elute DNA target by adding 22 μl of 10 mM Tris-HCl, pH 8.0 or 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature. Put the tube/PCR plate in the magnetic stand until the solution is clear.

3.7.10. Transfer 20 μl of supernatant (or desired volume) to a new tube/well, and proceed to bead based size selection.

 

3.8. PCR Amplification of Adaptor Ligated DNA

Use Option A for any NEBNext oligos kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes.

      Use Option B for any NEBNext oligo kits where index primers are supplied in 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined.

3.8.1A. Forward and Reverse Primers Not Already Combined
Mix the following components in sterile strip tubes:

Adaptor Ligated DNA Fragments (from Step 3.7.9) 15 µl
 (blue) Index Primer/i7 Primer*,** 5 µl
 (blue) Universal PCR Primer/i5 Primer*,** 5 µl 
 (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix
25 µl
Total Volume 50 µl

*NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.
**Use only one i7 primer/index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample.

3.8.1B. Forward and Reverse Primers Already Combined
Mix the following components in sterile strip tubes:

Adaptor Ligated DNA Fragments (from Step 3.7.9)  15 µl
 (blue) Index (X)/Universal Primer Mix*  10 µl
 (blue) NEBNext Ultra II Q5 Master Mix 25 µl
Total Volume 50 µl

*NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.

3.8.2. PCR cycling conditions:

CYCLE STEP TEMP TIME CYCLES
Initial Denaturation 98°C 30 seconds 1
Denaturation
Annealing/Extension
98°C
65°C
10 seconds
75 seconds
4–10*
Final Extension 65°C 5 minutes 1
Hold 4°C  

*We suggest 4-6 PCR cycles for 1 μg DNA input, 9–10 cycles for 50 ng. Further optimization of PCR cycle number may be required.

3.8.3. Proceed to cleanup of PCR Amplification (Section 3.9).

 

3.9. Cleanup of PCR Amplification

3.9.1. Vortex AMPure XP Beads to resuspend.

3.9.2. Add 45 μl (0.9X) of resuspended AMPure XP Beads to the PCR reactions (~50 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

3.9.3. Incubate for 5 minutes at room temperature.

3.9.4. Put the tube/ PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.

3.9.5. Add 200 μl of 80% ethanol to the PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

3.9.6. Repeat Step 3.9.5 once.

3.9.7. Air dry the beads for up to 5 minutes while the tube/PCR plate is 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.

3.9.8. Remove the tube/plate from the magnet. Elute DNA target from the beads into 30 μl of 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature. Put the tube/PCR plate in the magnetic stand until the solution is clear.

3.9.9. Transfer 25 μl of the supernatant to a clean LoBind tube. Libraries can be stored at –20°C.

3.9.10. Dilute 2–3 μl of the library 5 fold with 10 mM Tris or 0.1X TE, and assess the library quality on an Agilent Bioanalyzer (High Sensitivity Chip).

 

Figure 3.1: Example of a library prepared with normal human liver FFPE DNA.