Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370)

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This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input.
Colored bullets indicate the cap color of the reagent to be added

Starting Material: 5 ng–1 µg fragmented DNA.

1. NEBNext End Prep

1.1. Mix the following components in a sterile nuclease-free tube:

 (green) End Prep Enzyme Mix
3.0 μl
 (green) End Repair Reaction Buffer (10X)
6.5 μl
Fragmented DNA
55.5 μl
Total volume
65 μl

1.2. 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 the 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

 

2. Adaptor Ligation

If DNA input is < 100 ng, dilute the NEBNext Adaptor for Illumina (provided at 15 µM) 10 fold in 10 mM Tris-HCl with 10 mM NaCl to a final concentration of 1.5 µM, use immediately.

2.1. Add the following components directly to the End Prep reaction mixture and mix well:

(red) Blunt/TA Ligase Master Mix
15 µl
NEBNext Adaptor for Illumina*
 2.5 µl
Ligation Enhancer
1 µl
Total volume
83.5 µl

* The NEBNext adaptor is provided in the NEBNext oligos kit. NEB has several oligo kit options, which are supplied separately from the library prep kit.

Note: Ligation Enhancer and Blunt/TA Ligase Master Mix can be mixed ahead of time and is stable for at least 8 hours at 4°C. We do not recommend adding adaptor to a premix in the adaptor ligation step. For best results add adaptor last and mix well immediately or premix adaptor and sample and then add the other ligation reagents.

2.2. Set a 100 µl or 200 µl pipette to 80 µl and then pipette the entire volume up and down to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. Caution: The blunt/TA Ligase Master Mix is 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.

2.3. Incubate at 20°C for 15 minutes in a thermal cycler.

2.4. Add 3 μl of  USER™ Enzyme to the ligation mixture from Step 3.

Note: Steps 2.4 and 2.5 are only required for use with NEBNext Adaptors. USER Enzyme can be found in the NEBNext Singleplex or Multiplex Oligos for Illumina.

2.5. Mix well and incubate at 37°C for 15 minutes with the heated lid set to ≥ 47°C.

Safe Stopping Point: It is safe to store the library at -20°C.

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 size selection/cleanup of adaptor-ligated DNA. Once thawed, gently mix by inverting the tube several times.


3. Size Selection or Cleanup of Adaptor-ligated DNA

 Size selection is optional. If the starting material is > 50 ng, follow the protocol for size selection in Section 3A. For input less than 50 ng, size selection is not recommended. Follow the protocol for cleanup without size selection in Section 3B.

3A. Size Selection of Adaptor-ligated DNA

Note: The volumes of SPRIselect or AMPure XP reagent provided here are for use with the sample contained in the exact buffer at this step. These volumes may not work properly for a size selection at a different step in the workflow, or if this is a second size selection at this step. For size selection 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 a starting volume of 100 µl. Size selection conditions were optimized with AMPure XP beads; however, SPRIselect beads can be used following the same conditions.

To select a different insert size than 200 bp, please use the volumes in this table:

Table 1.1: Recommended conditions for bead based size selection.
LIBRARY
PARAMETERS
APPROXIMATE
INSERT SIZE
150 bp 200 bp 250 bp 300-400 bp 400-500 bp 500-700 bp
Total Library Size
(insert + adaptor)
270 bp 320 bp 400 bp 400-500 bp 500-600 bp 600-800 bp
VOLUME TO
BE ADDED (μl)
1st Bead Selection 65 55 45 40 35 30
2nd Bead Selection 25 25 25 20 15 15

3A.1. Vortex SPRIselect beads to resuspend. AMPure XP beads can be used as well. If using AMPure XP beads, please allow the beads to warm to room temperature for at least 30 minutes before use.

3A.2. Add 13.5 µl of dH2O to the ligation reaction for a 100 µl total volume.

3A.3. Add 55 µl (0.55X) of resuspended SPRIselect beads to the 100 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.4. Incubate samples on bench top for at least 5 minutes at room temperature.

3A.5. 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.6. 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.7. Add 25 µl (0.25X) resuspended SPRIselect 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.8. 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.9. 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.10. 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.11. Repeat Step 3A.10 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.12. 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.

3A.13. 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.14. 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.15. 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.

Safe Stopping Point: It is safe to store the library at -20°C.

3B. Cleanup of Adaptor-ligated DNA without Size Selection

Note: the volumes of SPRIselect or AMPure XP reagent provided here are for use with the sample contained in the exact buffer at this step. These 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 beads to resuspend (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.

3B.2. Add 86.5 µl (1X) resuspended SPRIselect 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 3B.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.

Use Option A for any NEBNext oligos kit where index primers are supplied in tubes. These kits have t he forward and reverse primers supplied in separate tubes.

       Use Option B for any NEBNext oligos kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined.

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

4.1A. Forward and Reverse Primer not already combined

Adaptor Ligated DNA Fragments
(Step 3A.15 or 3B.11)
15 µl
 (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix 25 µl
 (blue) Index Primer/i7 Primer*,** 5 µl
 (blue) Universal PCR Primer/i5 Primer*,** 5 µl
Total volume 50 µl

4.1B. Forward and Reverse Primer already combined

Adaptor Ligated DNA Fragments
(Step 3A.15 or 3B.11)
15 µl
 (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix 25 µl
 (blue) Index/Universal Primer* 10 µl
Total volume 50 µl

* NEBNext oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext oligo kit manual for determining valid barcode combinations.
** Use only one i7 primer/index primer per sample. Use only one i5 primer (or the universal primer for a single index kits) per sample.
 
4.2. 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.

4.3. Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:

CYCLE STEP TEMP TIME CYCLES
Initial Denaturation 98°C 30 seconds 1
Denaturation 98°C 10 seconds 4–12*
Annealing/Extension 65°C 75 seconds
Final Extension 65°C 5 minutes 1
Hold 4°C

* 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 your samples. 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).

Table 4.1.

INPUT DNA IN THE END PREP REACTION # OF CYCLES:
1 µg 4
50 ng 7-8
5 ng 12

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.

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

 

5. Cleanup of PCR Reaction

Note: the volumes of SPRIselect or AMPure XP reagent provided here are for use with the sample contained in the exact buffer at this step. 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 beads to resuspend. 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.

5.2. Add 45 µl (0.9X) resuspended SPRIselect 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 1: Examples of libraries prepared with human IMR-90 gDNA