Protocol for use with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645, E7103)

Symbols
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.
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, samples can be diluted with 10 mM Tris-HCl, pH 8.0 or 0.1X TE.

1.  NEBNext End Prep

1.1.     Add the following components to a sterile nuclease-free tube:

(green) NEBNext Ultra II End Prep Enzyme Mix
 3 μl
(green) NEBNext Ultra II End Prep Reaction Buffer  7 μl
 Fragmented DNA
 50 μl
 Total Volume
 60 μl

1.2.     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.

1.3.     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.

2.  Adaptor Ligation
2.1.     Determine whether adaptor dilution is necessary.
    If DNA input is < 100 ng, dilute the NEBNext Adaptor for Illumina in a solution of 10 mM Tris-HCI containing 10mM NaCl, pH 7.5.

    Table 2.1: 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
    Note: The appropriate adaptor dilution for your sample input and type may need to be optimized experimentally. The dilutions provided here are a general starting point. Excess adaptor should be removed prior to PCR enrichment.
2.2.     Add the following components directly to the End Prep Reaction Mixture:
 
End Prep Reaction Mixture (Step 1.3 in Section 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
 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, #E7710, #E7730 , #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 adding adaptor to a premix in the Adaptor Ligation Step.

2.3.     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).

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

2.5.     Add 3 μl of (red) USER™ Enzyme to the ligation mixture from Step 2.3.

Note: Steps 2.5 and 2.6 are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, and #E6609 ) Oligos for Illumina.

2.6.     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.


3.  Size Selection or Cleanup of Adaptor-ligated DNA

If the starting material is greater than 50 ng, follow the protocol for size selection in Section 3A. For input less than or equal to 50 ng, size selection is not recommended to maintain library complexity. Follow the protocol for cleanup without size selection in Section 3B.


3.A  Size Selection of Adaptor-ligated DNA
  Note: The following section is for cleanup of the ligation reaction. The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. 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. These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.


The following size selection protocol is for libraries with 200 bp inserts only. For libraries with different size fragment inserts, refer to the table below for the appropriate volumes of beads to be added. The size selection protocol is based on starting volume of 96.5 µl.
To select a different insert size than 200 bp, please use the volumes in this table:

Table 3.1: Recommended conditions for bead based size selection.

   APPROXIMATE
INSERT SIZE
 150 bp
 200 bp  250 bp
 300-400 bp
 400-500 bp
 500-700 bp
LIBRARY
PARAMETERS
 Approx. Final Library Size (Insert+Adaptor+primers)
270 bp
320 bp
370 bp
480
bp
600
bp
750-800 bp
 VOLUME TO
BE ADDED (µl)
 1st Bead Selection
50  40 30 25 20 15
 2nd Bead Selection
25 20 15 10 10 10

3A.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

3A.2. Add 40 μl (~ 0.4X) of resuspended beads to the 96.5 μl ligation reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

3A.3. Incubate samples on bench top for at least 5 minutes at room temperature.

3A.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.

3A.5. After 5 minutes (or when the solution is clear), 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.

3A.6. Add 20 μl (0.2X) resuspended SPRIselect or NEBNext Sample Purification Beads to the supernatant and mix at least 10 times. Be careful to expel all of the liquid from the tip during the last mix. Then incubate samples on the bench top for at least 5 minutes at room temperature.

3A.7. 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.

3A.8. After 5 minutes (or when the solution is clear), 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).

3A.9. Add 200 μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

3A.10. Repeat Step 3.A.9 once. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

3A.11. Air dry the beads for up to 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. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

3A.12. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads into 17 μl of 10 mM Tris-HCl or 0.1X TE.

3A.13. Mix well on a vortex mixer or by pipetting up and down 10 times. Incubate for at least 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 back on the magnetic stand.

3A.14. Place the tube/plate on a magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 μl to a new PCR tube for (amplification).

  Samples can be stored at -20°C.


3.B  Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)

The following section is for cleanup of the ligation reaction. If your input DNA is > 100 ng, follow the size selection protocol in section 3.A.

Note: The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. 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. These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.


3B.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

3B.2. Add 87 μl (0.9X) resuspended beads to the Adaptor Ligation reaction.
Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

3B.3. Incubate samples on bench top for at least 5 minutes at room temperature.

3B.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.

3B.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).

3B.6. Add 200 μl of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

3B.7. Repeat Step 3.B.6 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

3B.8. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

3B.9. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 μl of 10 mM Tris-HCl or 0.1X TE.

3B.10. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 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 back on the magnetic stand.

3B.11. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 μl to a new PCR tube.

Samples can be stored at -20°C.


4.  PCR Enrichment of Adaptor-ligated DNA

 Note:Check and verify that the concentration of your oligos is 10 μM.

Follow Section 4.1.1A if you are using the following oligos (10 μM primer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350)
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335)
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500)
NEBNext Multiplex Oligos for Illumina (Set 3, NEB #E7710)
NEBNext Multiplex Oligos for Illumina (Set 4, NEB #E7730)
NEBNext Multiplex Oligos for Illumina (Dual Index Primers, NEB #E7600)

Follow Section 4.1.1B if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)

4.1  PCR Amplification





4.1.1A.   Forward and Reverse Primer not already combined

Add the following components to a sterile strip tube:

Adaptor Ligated DNA Fragments (Step 3.1.14 or 3.2.11)
15 μl
(blue) NEBNext Ultra II Q5 Master Mix
 25 μl
(blue) Index Primer/i7 Primer*,**
 5 μl
(blue) Universal PCR Primer/i5 Primer*, ***
 5 μl
 Total volume
 50 μl
* The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #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, #E7710, #E7730) 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.

4.1.2.     Set a 100 μl or 200 μl pipette to 40 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.

4.1.3.     Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:
CYCLE STEP TEMPERATURE TIME CYCLES
Initial Denaturation 98°C 30 seconds 1
Denaturation
Annealing/Extension
98°C
65°C
10 seconds
75 seconds
3–15*
Final Extension 65°C 5 minutes 1
Hold 4°C  
*The number of PCR cycles should be chosen based on input amount and sample type. Thus, samples prepared with a different method prior to library prep may require re-optimization of the number of PCR cycles. The number of cycles should be high enough to provide sufficient library fragments for a successful sequencing run, but low enough to avoid PCR artifacts and over-cycling (high molecular weight fragments on Bioanalyzer). The number of PCR cycles recommended in Table 4.1 are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples. Use Table 4.2 for applications requiring high library yields (~1 µg) such as target enrichment.

4.1.1B.   Forward and Reverse Primer already combined

Add the following components to a sterile strip tube:

Adaptor Ligated DNA Fragments (Step 3.1.14 or 3.2.11)
 15 μl
(blue) NEBNext Ultra II Q5 Master Mix  25 μl
(blue) Index/Universal Primer****  10 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.
 
4.1.2.     Set a 100 μl or 200 μl pipette to 40 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.

4.1.3.     Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:
CYCLE STEP TEMPERATURE TIME CYCLES
Initial Denaturation 98°C 30 seconds 1
Denaturation
Annealing/Extension
98°C
65°C
10 seconds
75 seconds
3–15*
Final Extension 65°C 5 minutes 1
Hold 4°C  
*The number of PCR cycles should be chosen based on input amount and sample type. Thus, samples prepared with a different method prior to library prep may require re-optimization of the number of PCR cycles. The number of cycles should be high enough to provide sufficient library fragments for a successful sequencing run, but low enough to avoid PCR artifacts and over-cycling (high molecular weight fragments on Bioanalyzer). The number of PCR cycles recommended in Table 4.1 are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples. Use Table 4.2 for applications requiring high library yields (~1 µg) such as target enrichment.

Table 4.1.
INPUT DNA IN
THE END PREP
REACTION
# OF CYCLES REQUIRED FOR STANDARD LIBRARY PREP ~100 ng (30-100 nM):
 1 µg*
3**
500 ng* 3**
100 ng* 3
50 ng 3-4
10 ng 6-7
5 ng 7-8
1 ng 9-10
0.5 ng 10-11
*  These input ranges will work best when size selection is done
** NEBNext adaptors contain a unique truncated design. Libraries constructed with NEBNext adaptors require a minimum of 3 amplification cycles to add the complete adaptor sequences for downstream processes.


Table 4.2.
INPUT DNA IN
THE END PREP
REACTION
# OF CYCLES REQUIRED FOR TARGET ENRICHMENT LIBRARY PREP (~1 µg):
 1 µg*
3-4*,**
500 ng* 4-5*
100 ng* 6-7*
50 ng 7-8
10 ng 9-10
5 ng 10-11
1 ng 12-13
0.5 ng 14-15
*  Cycle number was determined for size selected libraries.
** NEBNext adaptors contain a unique truncated design. Libraries constructed with NEBNext adaptors require a minimum of 3 amplification cycles to add the complete adaptor sequences for downstream processes.


4.1.4. Proceed to Cleanup of PCR Amplification in Section 5.

5.  Cleanup of PCR Reaction

Note: The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. 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. These volumes may not work properly for a cleanup at a different step in the workflow. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.

5.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

5.2. Add 45 μl (0.9X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

5.3. Incubate samples on bench top for at least 5 minutes at room temperature.

5.4. 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.

5.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).

5.6. Add 200 μl of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

5.7. Repeat Step 5.6 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/ plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

5.8. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

5.9. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 μl of 0.1X TE.

5.10. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 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 back on the magnetic stand.

5.11. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 30 μl to a new PCR tube for and store at –20°C.

5.12. Check the size distribution on an Agilent Bioanalyzer High Sensitivity DNA chip. The sample may need to be diluted before loading.
 Samples can be stored at -20°C.
Figure 5.1: Examples of libraries prepared with human DNA (NA19240).