Protocol for use with NEBNext DNA Library Prep Reagent Set for Illumina (E6000)

1. Mix the following components in a sterile microfuge tube:
   Fragmented DNA 1-75 μl
    (green) Phosphorylation Reaction Buffer (10X) 10 μl
   (green) T4 DNA Polymerase 5 μl
   (green) T4 Polynucleotide Kinase 5 μl
   (green) dNTPs 4 μl
   ( green) DNA Polymerase I, Large (Klenow) 1 μl
   Sterile H₂O variable
   -----------------------------------------------
   Total volume 100 μl
2. Incubate in a thermal cycler for 30 minutes at 20°C.

1. Vortex AMPure XP beads to resuspend.
2. Add 160 μl (1.6X) 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 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 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 40 μl of 10 mM Tris-HCl or 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 32 μl of the supernatant to a fresh, sterile microfuge tube.

1. Mix the following components in a sterile microfuge tube:
   End Repaired, Blunt DNA 32 μl
    (yellow) NEBuffer 2 (10X) 5 μl
   (yellow) Deoxyadenosine 5´-Triphosphate 10 μl
   (yellow) Klenow Fragment (3´→ 5´ exo^–) 3 μl
   -----------------------------------------------
   Total volume 50 μl
2. Incubate in a thermal cycler for 30 minutes at 37°C.

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 the DNA targets.
5. 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.
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 the DNA target from the beads by adding 15 μl of 10 mM Tris-HCl or 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 10 μl of the supernatant to a fresh, sterile microfuge tube.

1. Mix the following components in a sterile microfuge tube:
   dA-Tailed DNA 10 μl
    (red) Quick Ligation Reaction Buffer (2X) 25 μl
    (red) NEBNext Adaptor* 10 μl
    (red) Quick T4 DNA Ligase 5 μl
   -----------------------------------------------
   Total volume 50 μl
   * The NEBNext adaptor can be found in NEBNext Singleplex (NEB #E7350 ) or Multiplex (NEB #E7335 , #E7500 , #E6609 or #E7600 ) Oligos for Illumina.


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.
   
   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 , #E6609 and #E7600 ) Oligos for Illumina.
   
   
4. Proceed to Cleanup Using AMPure XP Beads in Section 1.6
   
   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 90 μl 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 sample 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 the DNA target by adding 105 μl of 10 mM Tris-HCl 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 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 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 according to Table 1.1 above.

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

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 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 the 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 the DNA target from the beads by adding 17 μl of 10 mM Tris-HCl or 0.1X TE.
10. Mix well on a vortex mixer or by 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 15 μl of the supernatant to a clean PCR tube and proceed to enrichment.

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

Alternatively, adaptor ligated DNA can be size selected using a number of other methods including E-Gel SizeSelect Gels or standard 2% agarose gels. Purify DNA sample on one column and elute in 17 μl of sterile water or elution buffer.

1.8 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 1.8A 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 1.8B if you are using NEBNext Multiplex Oligos for Illumina
(96 Index Primers, NEB #E6609).
Follow Section 1.8C 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

1.8A PCR Enrichment of Adaptor Ligated DNA

1. Mix the following components in a sterile strip tubes:
   Adaptor Ligated DNA Fragments 15 μl
    (blue) Index Primer/i7 Primer*,** 5 μl
    (blue) Universal PCR Primer/i5 Primer*,*** 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 , #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 (NEB #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	98°C	10 seconds	4–8*
   Annealing	65°C	30 seconds
   Extension	72°C	30 seconds
   Final Extension	72°C	5 minutes	1
   	4°C	Hold

   *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 "Clean Up Using AMPure XP Beads" Section 1.9

1.8B PCR Enrichment of Adaptor Ligated DNA

1. Mix the following components in a 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 cobinations and tips for setting up PCR reactions.


2. PCR cycling conditions:

   CYCLE STEP	TEMP	TIME	CYCLES
   Initial Denaturation	98°C	30 seconds	1
   Denaturation	98°C	10 seconds	4–8*
   Annealing	65°C	30 seconds
   Extension	72°C	30 seconds
   Final Extension	72°C	5 minutes	1
   	4°C	Hold

   ** 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 a sterile strip tubes:
   Adaptor Ligated DNA Fragments 15 μl
    (blue) Index Primer*,** 2.5 μl
    (blue) Universal PCR Primer* 2.5 μl
    (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix 25 μl
   Sterile H2O 5 μl
   -----------------------------------------------
   Total volume 50 μl
   
   * 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.


2. PCR cycling conditions:

   CYCLE STEP	TEMP	TIME	CYCLES
   Initial Denaturation	98°C	30 seconds	1
   Denaturation	98°C	10 seconds	4–8*
   Annealing	65°C	30 seconds
   Extension	72°C	30 seconds
   Final Extension	72°C	5 minutes	1
   	4°C	Hold

   ** 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. Vortex Agencourt 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 plate from the magnet. Elute DNA target from the beads into 30 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 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-HCl 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 300–320 bp.