Protocol (E6040) for use with NEBNext Singleplex (#E7350) or Multiplex (#E7335, #E7500) Oligos for Illumina

Protocol

Starting Material: 1–5 μg of Fragmented DNA to 200 bp

End Repair of Fragmented DNA

1. Mix the following components in a sterile microfuge tube:
   Fragmented DNA   1–85 μl
   NEBNext End Repair Reaction Buffer (10X)   10 μl
   NEBNext End Repair Enzyme Mix   5 μl
   Sterile H₂O   variable
   ---------------------------------------------------------------------
   Total volume   100 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 160 μl (1.6X) 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 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 by adding 50 μl sterile water to the beads. 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. Without disturbing the bead pellet, carefully transfer 42 μl of the supernatant to a fresh, sterile microfuge tube.

Alternatively, adaptor ligated DNA can be purified on one purification column. Purify DNA sample on one QIAquick® (Qiagen) column and elute in 42 μl of sterile water or elution buffer.

dA-Tailing of End Repaired DNA

1. Mix the following components in a sterile microfuge tube:
   End Repaired, Blunt DNA   42 μl
   NEBNext dA-Tailing Reaction Buffer (10X)   5 μl
   Klenow Fragment (3´→ 5´ exo–)   3 μl
   -----------------------------------------------------------
   Total volume   50 μl
2. Incubate in a thermal cycler for 30 minutes at 37°C.

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 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 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 by adding 30 μl sterile water to the beads. 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. Without disturbing the bead pellet, carefully transfer 25 μl of the supernatant to a fresh, sterile microfuge tube.

Alternatively, adaptor ligated DNA can be purified on one purification column. Purify DNA sample on one MinElute column and elute in 25 μl of sterile water or elution buffer.

Adaptor Ligation of dA-Tailed DNA

1. Mix the following components in a sterile microfuge tube: 
   
   dA-Tailed DNA   25 μl
   Quick Ligation Reaction Buffer (5X)   10 μl
   NEBNext Adaptor*   10 μl
   Quick T4 DNA Ligase   5 μl
   ---------------------------------------------------------------------
   Total volume   50 μl 
   
   * Adaptors can be purchased separately under NEB #E7335, #E7350, #E7500.
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.

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 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 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
8. Elute DNA target by adding 105 μl sterile water to the beads for bead-based size selection as detailed In the next section, or at desired volume for size selection using E-Gel® (Life Technologies, Inc.) size select gels or standard 2% agarose gels. 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 (or desired volume) to a new tube/well, and proceed to bead based size selection.

Alternatively, adaptor ligated DNA can be purified on one purification column. Elute in sterile water in a volume desired for subsequent size selection.

Size Select Adaptor Ligated DNA Using AMPure XP Beads

Insert Size	150 bp	200 bp	250 bp	300 bp	400 bp	500 bp	700 bp
Total library size (insert + adaptor)	270 bp	320 bp	370 bp	420 bp	530 bp	660 bp	820 bp
Bead: DNA ratio* 1st bead selection	0.9X	0.8X	0.7X	0.6X	0.55X	0.5X	0.45X
Bead: DNA ratio* 2nd bead selection	0.2X	0.2X	0.2X	0.2X	0.15X	0.15X	0.15X

Table 1: Recommended Conditions for dual bead-based size selection.

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.

1. Add 80 μl (0.8X) 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 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.
7. Repeat Step 6 once.
8. Air dry beads for 10 minutes while the tube/PCR plate Is on the magnetic stand with the lid open.
9. Elute DNA target from the beads into 25 μl sterile 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.
   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 PCR Master Mix in the subsequent PCR step.
10. Without disturbing the bead pellet, carefully transfer 20 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 22 μl of sterile water or elution buffer.

PCR Enrichment Adaptor Ligated DNA

1. Mix the following components in a sterile microfuge tube: 
   DNA   20 μl
   Universal PCR Primer (25 μM)   2.5 μl
   Index Primer (1)* (25 μM)   2.5 μl
   NEBNext High-Fidelity 2X PCR Master Mix**   25 μl
   ---------------------------------------------------------------------
   Total volume   50 μl
   
   *Note: If you are using the NEBNext Multiplex Oligos for Illumina (Index Primers 1-12), 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	4–8*
   Final extension	72°C 4°C	5 min hold	1

   
   *If library construction was performed with 5 μg of starting material, use 4 cycles of amplification. If starting material was 1μg, use 6-8 cycles of amplification. However, optimization of PCR cycle number may be required to avoid over-amplification.

Clean Up Using AMPure XP Beads

1. Vortex AMPure XP 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 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 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
8. 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 put the tube/PCR plate in the magnetic stand until the solution is clear.
9. Without disturbing the bead pellet, carefully transfer 25 μl of the supernatant to a clean LoBind® (Eppendorf AG) tube, and store at -20°C.
   Alternatively, adaptor ligated DNA can be purified on one purification column. Purify DNA on one QIAQuick column and elute in 30 μl of 0.1X TE Buffer.
10. Dilute the library 20 fold with nuclease free water, 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 300–320 bp.