Nextera XT DNA Library Prep
Protocol Guide
For Research Use Only. Not for use in diagnostic procedures.
Tagment Genomic DNA
Amplify Libraries
Clean Up Libraries
Check Libraries
Normalize Libraries
Pool Libraries
Acronyms
Technical Assistance
ILLUMINA PROPRIETARY
Document # 1000000006564 v00
January 2016
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Preparation
1
Save the following tagmentation program on the thermal cycler:
} Choose the preheat lid option
} 55°C for 5 minutes
} Hold at 10°C
Procedure
1
Add the following items in the order listed to a new Hard-Shell skirted PCR plate.
Pipette to mix.
Item
TD
Normalized gDNA
Volume (µl)
10
5
2
Add 5 µl ATM. Pipette to mix.
3
Centrifuge at 280 × g at 20°C for 1 minute.
4
Place on the thermal cycler and run the tagmentation program.
5
Add 5 µl NT. Pipette to mix.
6
Centrifuge at 280 × g at 20°C for 1 minute.
7
Incubate at room temperature for 5 minutes.
8
[Optional] To assess tagmentation, run 1 µl sample on an Agilent Technology 2100
Bioanalyzer using a High Sensitivity DNA chip.
Nextera XT DNA Library Prep Protocol Guide
3
Tagment Genomic DNA
Tagment Genomic DNA
Amplify Libraries
Preparation
1
Save the following program on the thermal cycler:
} Choose the preheat lid option.
} 72°C for 3 minutes
} 95°C for 30 seconds
} 12 cycles of:
} 95°C for 10 seconds
} 55°C for 30 seconds
} 72°C for 30 seconds
} 72°C for 5 minutes
} Hold at 10°C
Procedure
1
[24 libraries] Arrange the index primers in the TruSeq Index Plate Fixture as follows.
} Arrange Index 1 (i7) adapters in columns 1–6 of the TruSeq Index Plate Fixture.
} Arrange Index 2 (i5) adapter in rows A–D of the TruSeq Index Plate Fixture.
2
[96 libraries] Arrange the index primers in the TruSeq Index Plate Fixture as follows.
} Arrange Index 1 (i7) adapters in columns 1–12 of the TruSeq Index Plate Fixture.
} Arrange Index 2 (i5) adapter in rows A–H of the TruSeq Index Plate Fixture.
3
Using a multichannel pipette, add 5 µl of each Index 1 (i7) adapter down each
column. Replace the cap on each i7 adapter tube with a new orange cap.
4
Using a multichannel pipette, add 5 µl of each Index 2 (i5) adapter across each row.
Replace the cap on each i5 adapter tube with a new white cap.
5
Add 15 µl NPM. Pipette to mix.
6
Centrifuge at 280 × g at 20°C for 1 minute.
7
Place on the thermal cycler and run the PCR program.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days. Alternatively,
leave on the thermal cycler overnight.
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Document # 1000000006564 v00
Preparation
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2
Prepare the following consumables:
Item
RSB
Storage
-25°C to 15°C
AMPure XP
Beads
2°C to 8°C
Instructions
Thaw at room temperature.
RSB can be stored at 2°C to 8°C after the initial thaw.
Let stand for 30 minutes to bring to room temperature.
Prepare fresh 80% ethanol from absolute ethanol.
Procedure
1
Centrifuge at 280 × g at 20°C for 1 minute.
2
Transfer 50 µl PCR product from the PCR plate to a new midi plate.
3
Add 30 µl AMPure XP beads.
4
Shake at 1800 rpm for 2 minutes.
5
Incubate at room temperature for 5 minutes.
6
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
7
Remove and discard all supernatant.
8
Wash 2 times with 200 µl 80% EtOH.
9
Using a 20 µl pipette, remove residual 80% EtOH.
10 Air-dry on the magnetic stand for 15 minutes.
11 Remove from the magnetic stand.
12 Add 52.5 µl RSB.
13 Shake at 1800 rpm for 2 minutes.
14 Incubate at room temperature for 2 minutes.
15 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
16 Transfer 50 µl supernatantto a new TCY plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.
Nextera XT DNA Library Prep Protocol Guide
5
Clean UpLibraries
Clean Up Libraries
Check Libraries
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[Optional] Run 1 µl of undiluted library on an Agilent Technology 2100 Bioanalyzer
using a High Sensitivity DNA chip.
Document # 1000000006564 v00
Normalize Libraries
Normalize Libraries
Preparation
1
Prepare the following consumables:
Item
LNA1
Storage
-25°C to -15°C
LNB1
2°C to 8°C
LNW1 2°C to 8°C
LNS1
Room
temperature
Instructions
Prepare under a fume hood.
Bring to room temperature. Use a 20°C to 25°C water bath as
needed.
Bring to room temperature. Use a 20°C to 25°C water bath as
needed.
Bring to room temperature. Use a 20°C to 25°C water bath as
needed.
Bring to room temperature.
Procedure
1
Transfer 20 µl supernatant to a new midi plate.
2
[96 samples] Add 4.4 ml LNA1 to a new 15 ml conical tube.
3
Thoroughly resuspend LNB1. Pipette to mix.
4
Transfer 800 µl LNB1 to the 15 ml conical tube. Invert to mix.
5
Pour the bead mixture into a trough and add 45 µl combined LNA1/LNB1.
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Shake at 1800 rpm for 30 minutes.
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Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
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Remove and discard all supernatant.
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Wash 2 times with 45 µl LNW1.
10 Add 30 µl 0.1 N NaOH.
11 Shake at 1800 rpm for 5 minutes.
12 During the 5 minute elution, label a new 96-well PCR plate SGP.
13 Add 30 µl LNS1 to the SGP plate. Set aside.
14 After the 5 minute elution, make sure that all samples are resuspended. Pipette to
mix.
15 Shake at 1800 rpm for 5 minutes.
16 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
17 Transfer the supernatant from the midi plate to the SGP plate.
18 Centrifuge at 1000 × g for 1 minute.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.
Nextera XT DNA Library Prep Protocol Guide
7
Pool Libraries
Preparation
1
To prepare for the sequencing run, begin thawing reagents according to the
instructions for your sequencing instrument.
2
If the SGP plate was stored frozen at -25°C to -15°C, thaw at room temperature.
Pipette to mix.
Procedure
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1
Centrifuge at 1000 × g at 20°C for 1 minute.
2
Transfer 5 µl from the SGP plate to a new PCR 8-tube strip.
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Label a new Eppendorf tube PAL.
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Transfer the contents of the PCR 8-tube strip to the PAL tube. Invert to mix.
5
Dilute pooled libraries to the loading concentration for the sequencing instrument
you are using. See the denature and dilute libraries guide for your instrument.
6
Store unused pooled libraries in the PAL tube and SGP plate at -25°C to -15°C for up
to 7 days.
Document # 1000000006564 v00
Acronyms
Acronyms
Acronym
Definition
ATM
Amplicon Tagment Mix
CAA
Clean Amplified Plate
CAN
Clean Amplified NTA Plate
LNA1
Library Normalization Additives 1
LNB1
Library Normalization Beads 1
LNS1
Library Normalization Storage Buffer 1
LNW1
Library Normalization Wash 1
LNP
Library Normalization Plate
NT
Neutralize Tagment Buffer
NPM
Nextera PCR Master Mix
NTA
Nextera XT Tagment Amplicon Plate
PAL
Pooled Amplicon Library
RSB
Resuspension Buffer
SGP
Storage Plate
TD
Tagment DNA Buffer
Nextera XT DNA Library Prep Protocol Guide
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Notes
For technical assistance, contact Illumina Technical Support.
Table 1 Illumina General Contact Information
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Nextera XT DNA Library Prep Protocol Guide
Technical Assistance
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Illumina
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