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  • Protocol for ChIP DNA (E7335)

    Introduction

    Starting Material: 10 ng of chromatin-immunoprecipitated (ChIP) qPCR verified or control DNA, in ≤ 40 μl of water or elution buffer
    End Repair and dA-Tailing Steps
    1. Perform end repair and dA-Tailing according to the appropriate protocol.
    Note: Dilute the NEBNext Adaptor (15 μM) to 1.5 μM in nuclease free water for immediate use.


    Protocol

    1. Adaptor Ligation of dA-Tailed DNA
      1. Mix the following components in a sterile microfuge tube:
        Reagent Set Protocol (NEB #E6200)
        dA-Tailed DNA   10 μl
        Quick Ligation Reaction Buffer (2X)   15 μl
        Diluted NEBNext Adaptor (1.5 μM)   1 μl
        Quick T4 DNA Ligase   4 μl
        --------------------------------------------------------------
        Total volume should be   30 μl

        Master Mix Protocol (NEB #E6240)
        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 should be   30 μl

      2. Incubate in a thermal cycler for 15 minutes at 20°C.
      3. Add 3 μl of USER enzyme, mix by pipetting up and down, and incubate at 37°C for 15 minutes

    2. Clean Up Using AMPure XP Beads
      1. Vortex AMPure XP beads to resuspend.
      2. Add 54 μl (1.8X) of resuspended AMPure XP beads to the ligation reaction (~ 30 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 105 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 the supernatant to a clean tube/PCR plate.

    3.  Alternately, adaptor ligated DNA can be purified on one purification column. Elute in sterile H2O in volume desired for subsequent size selection.

    4. Size Select Adaptor Ligated DNA Using AMPure XP Beads.

      Caution: The following size selection protocol is for libraries with 200 bp inserts only. For libraries with larger fragment inserts, please optimize bead:DNA ratio accordingly.

      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 25 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, adaptor ligated DNA can be size selected using a number of other methods including E-Gel size select gels or standard 2% agarose gels. Purify DNA sample on one column and elute in 25 μl of sterile water or elution buffer.

    5. PCR Enrichment of Adaptor Ligated DNA
      1. Mix the following components in a sterile microfuge tube:
        DNA   23 μl
        Universal PCR Primer (25 μM)   1 μl
        Index Primer (X)* (25 μM)   1 μl
        NEBNext High-Fidelity PCR Master Mix**   25 μl
        --------------------------------------------------------------
        Total volume   50 μl

        * Note: The NEBNext Multiplex Oligos for Illumina contains 12 PCR Primers, each with a
        different index. 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
        98°C 10 sec 15
        Annealing 65°C 30 sec
        Extension 72°C 30 sec
        Final Extension 72°C 5 min 1
        4°C hold
    6. 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 17 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). Check that the electropherogram shows a narrow distribution with a peak size around 275 bp is expected.