Protocol for use with NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (E6240)

   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.

1. Mix the following components in a sterile microfuge tube:
   ChIP DNA    1–40 μl
    (green) NEBNext End Repair Reaction Buffer (10X)   5 μl
    (green) 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.

1. Vortex AMPure XP beads to resuspend.
2. Add 90 μl (1.8X) of resuspended AMPure XP Beads to the reaction. Mix thoroughly ona 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% freshly prepared ethanol to the tube/PCR 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 from beads into 50 μl of 0.1X TE. 
9. Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature.
10. Put the tube/PCR plate in the magnetic stand until the solution is clear. Without disturbing the bead pellet, carefully transfer 44 μl of the supernatant to a fresh, sterile microfuge tube. 

1. Mix the following components in a sterile tube:
   End Repaired, Blunt DNA 44 μl
    (yellow) NEBNext dA-Tailing Reaction Buffer (10X) 5 μl
    (yellow) Klenow Fragment (3´→ 5´ exo^–) 1 μl
   -----------------------------------------------
   Total volume 50 μl
2. Incubate in a thermal cycler for 37°C for 30 minutes.

1. Vortex AMPure XP Beads to resuspend.
2. Add 90 μl (1.8X) of resuspended AMPure XP Beads to the 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 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 from beads into 25 μl of 0.1X TE. 
9. Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature. 

Dilute the  (red) NEBNext Adaptor for Illumina* (15 μM) 10-fold in 10 mM Tris-HCI or 10mM Tris-HCI with 10 mM NaCl to a final concentration of 1.5 μM.

1. Mix the following components in a sterile microfuge tube. 
   dA-Tailed DNA 19 μl
    (red) Quick Ligation Reaction Buffer (5X) 6 μl
   Diluted NEBNext Adaptor (1.5 μM) 1 μl
    (red) Quick T4 DNA Ligase 4 μl
   -----------------------------------------------
   Total volume 30 μl
2. Incubate in a thermal cycler for 15 minutes at 20°C. 
3. Add 3 μl of  USER enzyme, mix by gently pipetting up and down, and incubate at 37°C for 15 minutes.

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

1. Vortex AMPure XP Beads to resuspend
2. Add 54 μl of resuspended AMPure XP Beads to the ligation 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 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 from beads into 105 μl of 10 mM Tris-HCI or 0.1 X TE to the beads for bead-based size selection. Note: For size selection using E-Gel size select gels or standard 2% agarose gels, elute the DNA target at desired volume. 
9. Mix well on a vortex mixer or by pipetting up and down several times, and incubate for 2 minutes at room temperature. 
10. Put the tube/PCR plate in the magnetic stand until the solution is clear. Transfer 100 μl of supernatant (or desired volume) to a new tube/well, and proceed to bead based size selection.

1.7 Size Selection of Adaptor Ligated DNA Using Agencourt AMPure XP Beads

   The following size selection protocol is for libraries with 150 bp inserts only. For libraries with different size  fragment inserts, please optimize bead: DNA ration according to Table 1.1 above.

1. Add 90 μl (0.9X) resuspended AMPure XP Beads to 100 μl DNA solution. Mix well on a vortex mixer or by gently 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 sample 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 5 minutes while the tube/PCR plate is on the magnetic stand with lid open. Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
9. Remove the tube/plate from the magnet. Elute DNA target from the beads by adding 22 μl of 10 mM Tris-HCI or 0.1X TE. 
10. Mix well on a vortex mixer or by gently pipetting up and down and incubate for 2 minutes at room temperature.
11. Put the tube/PCR plate in the magnetic stand until the solution is clear. Without disturbing the bead pellet, carefully transfer 20 μl of the supernatant to a clean PCR tube and proceed to enrichment. 

  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. 

1. Mix the following components in sterile strip tubes:
Adaptor Ligated DNA Fragments 20 μl

  (blue) Index Primer/ i7 Primer*,** 2.5 μl

  (blue) Universal PCR Primer/ i5 Primer*,*** 2.5 μl

  (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix 25 μl

--------------------------------------------------------------------------------

Total volume 50 μl





* The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500 or #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 PCR reactions. 

** For use with NEBNext Multiplex Oligos (#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	10 seconds 75 seconds	15
   Final Extension	65°C	5 minutes	1
   Hold	4°C	∞

   
   

3. Proceed to Cleanup Ampure XP Beads in Section 1.9.

1. Mix the following components in sterile strip tubes:
Adaptor Ligated DNA Fragments 15 μl
 (blue) Index/ Universal Primer Mix* 10 μl
 (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix 25 μl
--------------------------------------------------------------------------------
Total volume 50 μl
* The primers are provided in NEBNext Multiplex Oligos for Illumina, NEB #E6609. Please refer to the NEB #E6609 manual for valid barcode combinations and tips for setting up PCR reactions. 


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	2-4*
Final Extension	65°C	5 minutes	1
Hold	4°C	∞

* If library construction was performed with 5 μg of starting material, use 2-3 cycles of amplification. If starting material was 1 μg, use 4 cycles of amplification. However, optimization of PCR cycle number may be required to avoid over-amplification.


3.  Proceed to Cleanup Using Ampure XP Beads in Section 1.9.

1. Mix the following components in sterile strip tubes:

* 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. 


1. 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	15
Final Extension	65°C	5 minutes	1
Hold	4°C	∞



2. Proceed to Cleanup Using Ampure XP Beads in Section 1.9.

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 the DNA target from the beads by adding 30 μl of 0.1X TE.
9. Mix well on a vortex mixer or by pipetting up an down and incubate for 2 minutes at room temperature.
10. Put the tube/PCR plate in the magnetic stand until the solution is clear. Without disturbing the bead pellet, carefully transfer 25 μl of the supernatant to a clean LoBind^®(Eppendorf AG) tube. Libraries can be stored at -20°C. 
11. Dilute 2-3 μl of the library 20 fold with 10 mM Tris-HCI or 0.1X TE and assess the library quality on a Bioanalyzer^®(Agilent Technologies, Inc.) high sensitivity chip. Check that the electropherogram shows a narrow distribution with a peak size approximately 275 bp.

Figure 1.1: Bioanalyzer traces of final library.