Protocol for use with NEBNext Ultra II DNA Library Prep with Sample Purification Beads (E7103)

Symbols
Stopping points in the protocol.
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

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.    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

2.    Set a 100 µl or 200 µl pipette to 50 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.

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
 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 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, #E7710, #E7730, #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.
 

Table 1.3A: 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
 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 Sample Purification Beads to resuspend.

2.    Add 40 μl of resuspended Sample Purification 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 Sample Purification 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 Sample Purification Beads to resuspend.

2.    Add 87 μl resuspended Sample Purification 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
 
Follow Section 1.4A 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 1.4B if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)

1.4A    PCR Amplification

1.    Add the following components to a sterile strip tube:


 Adaptor Ligated DNA Fragments (Step 11 in Section 1.3A or 1.3B)  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, #E7500, #E7710 or #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.

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.

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  
   
  *Follow the recommendations listed in Table 1.4 on page 9.

4.    Proceed to Cleanup of PCR Amplification in Section 1.5.

1.4B    PCR Amplification for use with NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)

1.    Add the following components to a sterile strip tube:

Adaptor Ligated DNA Fragments (Step 11 in Section 1.3A or 1.3B) 15 µl
(blue) Index/ Universal Primer Mix* 10 µl
(blue) NEBNext Ultra II Q5 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.    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.

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  

    *Follow the recommendations listed in Table 1.4 on page 9.

4.    Proceed to Cleanup of PCR Amplification in Section 1.5
   
Table 1.4

INPUT DNA IN
THE END PREP
REACTION
 # OF CYCLES REQUIRED TO
GENERATE A LIBRARY YIELD OF:
 INPUT  100 ng
 1 µg
 1 µg
 0*  3-4**
500 ng 0* 4-5**
100 ng 2-3 7-8**
50 ng 3-4 6-7
10 ng 6-7 9-10
5 ng 7-8 10-11
1 ng 9-10 12-13
0.5 ng 10-11 14-15


    * NEBNext adaptors contain a unique truncated design. Libraries constructed
    with NEBNext adaptors require a minimum of 2–3 amplification cycles to add
    the complete adaptor sequences for upstream processes.
**    Cycle number was determined for size selected libraries.

1.5    Cleanup of PCR Amplification

1.    Vortex Sample Purification Beads to resuspend.

2.    Add 45 μl of resuspended Sample Purification Beads to the PCR reactions (~ 50 μl). 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% 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 DNA target from beads into 33 μl 0.1X TE. Mix well by pipetting up and down at least 10 times. If necessary, quick spin the tube/plate to collect the liquid and incubate at room temperature for 2 minutes.

9.    Place the sample on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer 30 μl supernatant to a new PCR tube. Libraries can be stored at –20°C.

10.    Dilute 1 µl of the library 5 fold with 10 mM Tris-HCl or 0.1X TE and check the size distribution on an Agilent Bioanalyzer® (high sensitivity chip).

Figure 1.1: Examples of libraries prepared with human DNA (NA19240).

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