Protocol for use with NEBNext DNA Library Prep Master Mix Set for Illumina (NEB #E6040) and NEBNext High-Fidelity 2X PCR Master Mix (NEB #E6013)

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

2.1 NEBNext FFPE Repair

  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
  2. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.
  3. Incubate at 20°C for 15 minutes.
2.2 End Repair of Fragmented DNA

  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 H2O 29.5 μl
    Repaired DNA from above (Section 2.1) 62 μl
    --------------------------------------------------------------------------------------------
    Total volume 100 μl
  2. Incubate in a thermal cycler for 30 minutes at 20°C.
2.3 Cleanup Using AMPure XP Beads

  1. Vortex AMPure XP Beads to resuspend.
  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. Incubate for 5 minutes at room temperature.
  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.
  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.
  6. Repeat Step 5 once.
  7. Air dry beads for 5 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  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.
  9. Without disturbing the bead pellet, carefully transfer 42 μl of the supernatant to a fresh, sterile microfuge tube.
2.4 dA-Tailing of End Repaired DNA

  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
  2. Incubate in a thermal cycler for 30 minutes at 37°C.
2.5 Cleanup Using Ampure XP Beads

  1. Vortex AMPure XP Beads to resuspend.
  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. Incubate for 5 minutes at room temperature.
  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.
  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.
  6. Repeat Step 5 once.
  7. Air dry beads for 5 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  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.
  9. Without disturbing the bead pellet, carefully transfer 32.5 μl of the supernatant to a fresh, sterile microfuge tube.
2.6 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.

  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
    **Adaptors can be purchased separately under NEB #E7335, #E7500, #E7350, #E7600.
  2. Incubate in a thermal cycler for 15 minutes at 20°C.
  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 (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7600) Oligos for Illumina.
2.7 Cleanup of Adaptor-ligated DNA

  1. Vortex AMPure XP Beads to resuspend
  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. Incubate for 5 minutes at room temperature.
  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.
  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.
  6. Repeat Step 5 once.
  7. Air dry beads for 5 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  8. Remove the tube/plate from the magnet. Elute DNA target by adding 25 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.
  9. Transfer 20 μl of supernatant (or desired volume) to a new tube/well, and proceed to bead based size selection.
    Note: Be sure not to transfer any beads. Trace amounts of bead carryover may affect the optimal performance of the polymerase used in the NEBNext High-Fidelity 2X PCR Master Mix in the subsequent PCR step.

2.8 PCR Amplification of Adaptor Ligated DNA

Note: NEBNext Singleplex and Multiplex Oligos for Illumina (NEB #E7350, #E7335 and #E7500) now have new primer concentrations (10 μM). Please check oligo kit lot numbers to determine how to set up your PCR reaction.

Follow Section 2.8A if you are using the following oligos (10 μMprimer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350) lot 0071412
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) lot 0091412
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500) lot 0071412
NEBNext Multiplex Oligos for Illumina (Dual Index Primers, NEB #E7600) all lots

Follow Section 2.8B if you are using the following oligos (25 μM primer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350) lots 0051402 or 0061410
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) lots 0071402 or 0081407
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500) lots 0051402 or 0061407

2.8A PCR Amplification

  1. Mix the following components in sterile strip tubes:
    Adaptor Ligated DNA Fragments (from Step 9, Section 2.7) 20 μl
    (blue) Index Primer/i7 Primer*,** 2.5 μl
    (blue) Universal PCR Primer/i5 Primer*,*** 2.5 μl
    (blue) NEBNext High-Fidelity 2X PCR Master Mix 25 μl
    --------------------------------------------------------------------------------------------
    Total volume 50 μl
    * The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7600) Oligos for Illumina. For use with Dual Index Primers (NEB #E7600), look at the NEB #E7600 manual for valid barcode combinations and tips for setting up the PCR reaction.
    ** For use with NEBNext Multiplex Oligos (NEB #E7335 or #E7500) use only one Index Primer per PCR reaction. For use with Dual Index Primers (NEB #E7600) use only one i7 Primer per reaction.
    *** For use with Dual Index Primers (NEB #E7600) use only one i5 Primer per reaction.
  2. PCR cycling conditions:
    CYCLE STEP TEMP TIME CYCLES
    Initial Denaturation 98°C 30 seconds 1
    Denaturation
    Annealing
    Extension
    98°C
    65°C
    72 °C
    10 seconds
    30 seconds
    30 seconds
    4–10*
    Final Extension 72°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. Proceed to Cleanup Using Ampure XP Beads in Section 2.9

2.8B PCR Enrichment of Adaptor Ligated DNA for use with NEBNext Oligos NEB #E7600.

  1. Mix the following components in sterile strip tubes:
    Adaptor Ligated DNA Fragments (from Step 9, Section 2.7) 20 μl
    (blue) Index Primer*,** 1 μl
    (blue) Universal PCR Primer* 1 μl
    (blue) NEBNext High-Fidelity 2X PCR Master Mix 25 μl
    Sterile H2O 3 μl
    --------------------------------------------------------------------------------------------
    Total volume 50 μl
    * The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335 or #E7500) Oligos for Illumina.
    ** For use with NEBNext Multiplex Oligos (NEB #E7335 or #E7500) use only one Index Primer per PCR reaction.
  2. PCR cycling conditions:
    CYCLE STEP TEMP TIME CYCLES
    Initial Denaturation 98°C 30 seconds 1
    Denaturation
    Annealing
    Extension
    98°C
    65°C
    72 °C
    10 seconds
    30 seconds
    30 seconds
    4–10*
    Final Extension 72°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.

4.9 Cleanup Using AMPure XP Beads

  1. Vortex AMPure XP Beads to resuspend.
  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. Incubate for 5 minutes at room temperature.
  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.
  5. Add 200 μl of freshly prepared 80% 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.
  6. Repeat Step 5 once.
  7. Air dry the beads for 5 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  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.
  9. Transfer 25 μl of the supernatant to a clean LoBind tube. Libraries can be stored at –20°C.
  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 2.1: Example of a library prepared with normal human liver FFPE DNA.