Protocol for use with NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (E6200)

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Stopping points in the protocol.

Starting Material: 500 pg–1 μg fragmented DNA. We recommend that DNA be sheared in 1X TE. If the DNA volume post shearing is less than 50 μl, add 1X TE to a final volume of 50 μl. Alternatively, 10 mM Tris-HCl, pH 8.0 or 0.1X TE can be used.

1.1 NEBNext End Prep
  1. Mix the following components in a sterile nuclease-free tube:
     (green) NEBNext Ultra II End Prep Enzyme Mix 3 μl
     (green) NEBNext Ultra II End Prep Reaction Buffer 7μl
    Fragmented DNA 50 μl
    -----------------------------------------------
    Total volume 60 μl

  2. Set a 100 μl or 200 μl pipette to 50 μl and then gently pipette the entire volume up and down at least 10 times to mix throughly. Perform a quick spin to collect all liquid from the sides of the tube.

    Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.

  3. Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:
    30 minutes @ 20°C
    30 minutes @ 65°C
    Hold at 4°C

    If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.

1.2 Adaptor Ligation

If DNA input is ≤ 100 ng, dilute the NEBNext Adaptor for Illumina in 10 mM Tris-HCl or 10 mM Tris-HCl with 10 mM NaCl as indicated in Table 1.2.

Table 1.2: Adaptor Dilution
INPUT ADAPTOR DILUTION
(VOLUME OF ADAPTOR: TOTAL VOLUME)
WORKING ADAPTOR
CONCENTRATION
1 μg–101 ng No Dilution 15 μM
100 ng–5 ng 10-Fold (1:10) 1.5 μM
less than 5 ng 25-Fold (1:25) 0.6 μM
  1. Add the following components directly to the End Prep Reaction Mixture:
    End Prep Reaction Mixture (Step 3 in Section 1.1) 60 μl
     (red) NEBNext Ultra II Ligation Master Mix* 30 μl
     (red) NEBNext Ligation Enhancer 1 μl
     (red) NEBNext Adaptor for Illumina** 2.5 μl
    Fragmented DNA 50 μl
    -----------------------------------------------
    Total volume 93.5 μl

    * Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.
    ** The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7600, #E7535 and #E6609) Oligos for Illumina.

    Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend premixing the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step.

  2. Set a 100 μl or 200 μl pipette to 80 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
    (Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).

  3. Incubate at 20°C for 15 minutes in a thermocycler with the heated lid off.

  4. Add 3 μl of (red) USER™ Enzyme to the ligation mixture from Step 3.

    Note: This step is only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7600 and #E6609) Oligos for Illumina.

  5. Mix well and incubate at 37°C for 15 minutes with the heated lid set to ≥ 47°C.

    Samples can be stored overnight at –20°C.
1.3 Size Selection or Cleanup of Adaptor-ligated DNA
Size selection is optional. If the starting material is greater than 50 ng, follow the protocol for size selection in Section 1.3A to maintain library complexity. For input less than or equal to 50 ng, size selection is not recommended. *Follow the protocol for cleanup without size selection in Section 1.3B.

*If performing ChIP-seq, we recommend performing a size selection.
1.3A Size Selection of Adaptor Ligated DNA (for input > 50 ng or for ChIP-seq libraries)
The following size selection protocol is for libraries with 200 bp inserts only. For libraries with different size fragment inserts, refer to Table 1.3A for the appropriate volume of beads to be added. The size selection protocol is based on a starting volume of 96.5 μl. Size selection conditions were optimized with SPRIselect Beads; however, AMPure XP Beads can be used following the same conditions. If using AMPure XP beads, please allow the beads to warm to room temperature for at least 30 minutes before use.
Table 1.3A: Recommended conditions for bead based size selection.

LIBRARY
PARAMETERS
APPROXIMATE
INSERT SIZE
150 bp bp 200 bp 250 bp 300-400 bp 400-500 bp 500-700 bp
Total Library Size
(insert + adaptor + primers)
270 bp 320 bp 420 bp 520 bp 650 bp 700-800 bp
VOLUME TO
BE ADDED (μl)
1st Bead Selection 50 40 30 25 20 15
2nd Bead Selection 25 20 15 10 10 10

  1. Vortex SPRIselect Beads to resuspend.

  2. Add 40 μl of resuspended SPRIselect Beads to the 96.5 μl ligation reaction. Mix well by pipetting up and down at least 10 times.

  3. Incubate for 5 minutes at room temperature.

  4. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand. After the solution is clear (about 5 minutes), carefully transfer the supernatant containing your DNA to a new tube (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.

  5. Add 20 μl resuspended SPRIselect Beads to the supernatant, mix well and incubate for 5 minutes at room temperature.

  6. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA targets (Caution: do not discard beads).

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

  8. Repeat Step 7 once.

  9. Air dry the beads for 5 minutes while the tube/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.

  10. Remove the tube/plate from the magnet. Elute the DNA target from the beads into 17 μl of 10 mM Tris-HCl or 0.1 X TE. Mix well on a vortex mixer or by pipetting up and down. Incubate for 2 minutes at room temperature.

  11. Place the tube/plate on a magnetic stand. After the solution is clear (about 5 minutes), transfer 15 μl to a new PCR tube for amplification.

  12. Proceed to PCR Amplification in Section 1.4.

1.3B Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
  1. Vortex SPRIselect Beads to resuspend (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.

  2. Add 87 μl resuspended SPRIselect Beads to the ligation reaction. Mix well by pipetting up and down at least 10 times.

  3. Incubate for 5 minutes at room temperature.

  4. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand. 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).

  5. Add 200 μl of 80% freshly prepared ethanol to the tube 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 for a total of two washes.

  7. Air dry the beads for 5 minutes while the tube/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 the DNA target from the beads by adding 17 μl of 10 mM Tris-HCl or 0.1X TE.

  9. Mix well by pipetting up and down, or on a vortex mixer. Incubate for 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

  10. Place the tube/plate on the magnetic stand.

  11. After the solution is clear (about 5 minutes), transfer 15 μl to a new PCR tube for amplification.

1.4 PCR Enrichment 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 1.4A if you are using the following oligos (10 μM primer):
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 1.4B if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609) Follow Section 1.4C 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