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  • Protocol (E6240) for Use With NEBNext Singleplex (#E7350) or Multiplex (#E7335, #E7500) Oligos for Illumina

    Protocol

    Starting Material: 10 ng of chromatin-immunoprecipitated (ChIP) qPCR verified or control DNA, in < 40 μl of water or elution buffer

    End Repair of ChIP DNA
    1. In a sterile microfuge tube mix the following components:
      ChIP DNA   1–40 μl
      NEBNext End Repair Reaction Buffer  5 μl
      NEBNext End Repair Enzyme Mix   1 μl
      Sterile H2O   variable
      ---------------------------------------------------------------------
      Total volume   50 μl
    2. Incubate in a thermal cycler for 30 minutes at 20°C.
    Clean Up Using AMPure XP® Beads (Beckman Coulter, Inc.) 
    1. Vortex AMPure XP beads to resuspend.
    2. Add 90 μl (1.8X) of resuspended AMPure XP beads to the ligation reaction (~50 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 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    8. Elute DNA target from beads into 50 μl of 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the magnetic stand until the solution is clear.
    9. Transfer 44 μl of the supernatant to a clean LoBind® tube (Eppendorf AG), and store at -20°C.
    Alternatively, purify DNA sample on one purification column and elute in 44 μl of sterile dH2O or elution buffer.

    dA-Tailing of End Repaired DNA
    1. Mix the following components in a sterile microfuge tube:
      End Repaired DNA   44 μl
      NEBNext dA-Tailing Reaction Buffer (10X)   5 μl
      Klenow Fragment (3´→ 5´ exo)   1 μl
      -----------------------------------------------------------
      Total volume   50 μl
    2. Incubate at 37°C for 30 minutes.
    Clean Up 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 (~ 50 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 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    8. Elute DNA target from beads into 25 μl of 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the magnetic stand until the solution is clear.
    9. Transfer 19 μl of the supernatant to a clean LoBind tube, and store at -20°C.
    Alternatively, purify DNA sample on one purification column and elute in 19 μl of sterile dH2O or elution buffer.

    Adaptor Ligation of dA-Tailed DNA
    1. Mix the following components in a sterile microfuge tube: 

      End Repaired, dA-Tailed DNA   19 μl
      Quick Ligation Reaction Buffer (5X)   6 μl
      Diluted NEBNext Adaptor (1.5 μM)  1 μl
      Quick T4 DNA Ligase   4 μl
      ---------------------------------------------------------------------
      Total volume   30 μl 

    2. Incubate at 20°C for 15 minutes.
    3. Add 3 μl of USER™ enzyme mix by pipetting up and down, and incubate at 37°C for 15 minutes.
    Clean Up Using AMPure XP Beads
    1. Vortex AMPure XP beads to resuspend.
    2. Add 54 μl of resuspended AMPure XP beads to the ligation reaction (~30 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 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    8. Elute DNA target from beads into 110 μl of dH2O. Mix well on a vortex mixer or by pipetting up and down, and put the tube/pcr plate in the magnetic stand until the solution is clear.
    9. Transfer 100 μl of supernatant to a clean tube/PCR plate.
    Alternatively, purify DNA sample on one purification column and elute in 20 μl of sterile H2O or elution buffer.


    Size Selection of Adaptor Ligated DNA Using AMPure XP Beads


    Note: (X) refers to original sample volume of 100 μl.

    1. Add 90 μl (0.9x) resuspended AMPure xP beads to 100 μl DNA solution. Mix well on a vortex mixer or by pipetting up and down at least 10 times.

    2. Incubate for 5 minutes at room temperature.

    3. Place the tube/PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube/well (Caution: do not discard the supernatant). Discard beads that contain the large fragments.

    4. Add 20 μl (0.2x) resuspended AMPure xP beads to the supernatant, mix well and incubate for 5 minutes at room temperature.

    5. 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 DNA targets (Caution: do not discard beads).

    6. 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.

    7. Repeat Step 6 once.

    8. Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with lid open.

    9. Elute DNA target from beads into 30 μl water or 0.1x tE buffer. Mix well on a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the magnetic stand until the solution is clear.

    10. Transfer 23 μl of the supernatant to a clean PCR tube and proceed to enrichment.

    Alternatively, size selection can be performed using a number of methods including E-Gel® (Life Technologies, Inc.) size select gels or standard 2% agarose gels. NEB's 100 bp ladder (NEB #N3231) can be used to determine the size of the fragments. Isolate library fragments in the 175–225 base pair range. Purify the DNA on one purification column and elute in 25 μl of sterile water or elution buffer.

    Note: Be sure not to transfer any beads. Trace amounts of bead carry-over may affect the optimal performance of the polymerase used in the NEBNext High-Fidelity 2X PCR Master Mix in the subsequent PCR step.

    PCR Enrichment of Adaptor Ligated DNA
    1. Mix the following components in a sterile microfuge tube:
      Adaptor ligated DNA   23 μl
      NEBNext High-Fidelity 2X PCR Master Mix**  25 μl
      Universal PCR Primer (25 μM)   1 μl
      Index 1 Primer* (25 μM)   1 μL
      ---------------------------------------------------------------------
      Total volume   50 μl
      * If you are using the NEBNext Multiplex Oligos for Illumina (E#7335, #E7500), for each reaction, only one of the 12 PCR primer indices is used during the PCR step.
      ** NEBNext High-Fidelity 2X PCR Master Mix will be replacing Phusion High-Fidelity PCR Master Mix. Both vials will be supplied for a limited time only.
    2. PCR cycling conditions:
       Cycle Step
      Temp
      Time
      Cycles
      Initial Denaturation
       98°C
       30 sec
      1
      Denaturation
      Annealing
      Extension
       98°C
       65°C
       72°C
       10 sec
       30 sec
       30 sec
       15
       Final Extension
       72°C
         4°C
         5 min
           hold
       1

    Clean Up Using AMPure XP Beads

    1. Vortex beads to resuspend.

    2. Add 50 μl (1x) 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 DNA targets.

    5. Add 200 μl of 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 beads for 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.

    8. Elute DNA target from beads into 20 μl of 0.1x tE. Mix well on a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the magnetic stand until the solution is clear.

    9. transfer 15 μl of the supernatant to a clean LoBind tube, and store at –20°C.

    Alternatively, purify sample on one purification column and elute in 15 μl of sterile water or elution buffer.

    Dilute the library 20 fold with nuclease free water, and assess the library quality on a Bioanalyzer® (Agilent high sensitivity chip) (Agilent technologies, Inc.). Check that the electropherogram shows a narrow distribution with a peak size around 275 bp is expected (an example is shown below).