TruSeq Nano DNA Library Prep for
NeoPrep
Protocol Guide
For Research Use Only. Not for use in diagnostic procedures.
Fragment DNA
Prepare Samples for Loading
Set Up Run and Load Library Card
Unload Libraries
[Optional] Validate Libraries Manually
[Optional] Normalize Libraries Manually
Pool Libraries
Acronyms
Technical Assistance
ILLUMINA PROPRIETARY
Document # 15059579 v01
October 2015
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This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the
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document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed,
or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license
under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order
to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read
and understood prior to using such product(s).
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MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND
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ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)
DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).
© 2015 Illumina, Inc. All rights reserved.
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AMPure®, Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc.
Preparation
1
Turn on the Covaris instrument and follow manufacturer instructions to set up your
instrument.
Procedure
1
Quantify gDNA samples using a fluorometric-based method that uses dsDNA
binding dyes such as Qubit or QuantiFlour.
2
Normalize gDNA with RSB in a final volume of 15 µl in separate wells of a new
PCR plate:
} For a 350 bp insert size—1.67 ng/µl of sample
} For a 550 bp insert size—5 ng/µl of sample
3
Transfer 15 µl of each normalized DNA to a microTUBE-15.
4
Centrifuge, using a microTUBE adapter, at 3000 × g for 1 minute.
5
Fragment the DNA on a Covaris.
Table 1 Covaris S220 or E220 Settings
Setting
Duty factor
Peak Incident Power
Cycles per burst
Duration
Temperature
Water Level—S220
Water Level—E220
350 bp Insert
550 bp Insert
20%
18 W
50
45 seconds
22 seconds
20°C
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Table 2 Covaris M220 Settings
Setting
Duty factor
Peak Incident Power
Cycles per burst
Duration
Temperature
350 bp Insert
550 bp Insert
20%
30 W
50
42 seconds
23 seconds
20°C
6
Centrifuge, using a microTUBE adapter, at 600 × g for 5 seconds.
7
Transfer 15 µl fragmented DNA from each microTUBE-15 to a separate well of a new
PCR plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.
TruSeq Nano DNA Library Prep for NeoPrep Protocol Guide
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Fragment DNA
Fragment DNA
Prepare Samples for Loading
Procedure
4
1
Add 1 of the following to a new 1.5 ml microcentrifuge tube:
} For a 350 bp insert size—900 µl SC350
} For a 550 bp insert size—700 µl SC550
2
Vortex DMB until well-dispersed.
3
Add 100 µl DMB to the microcentrifuge tube containing SC350 or SC550. Vortex for
5 seconds.
4
Pour the DMB and SC mixture into a new reagent reservoir.
5
Add 35 µl DMB and SC mixture to each well of the sample plate. Pipette to mix.
6
Shake or vortex at 1400 rpm for 12 minutes.
Document # 15059579 v01
Procedure
1
Vortex the reagent plate for 3 seconds.
2
Centrifuge at 600 × g for 5 seconds.
3
Select Prepare Libraries on the NeoPrep System Welcome screen.
4
Do the following and then select Next.
} If running in BaseSpace mode, select a run.
} If running in standalone mode, use the following options to select a protocol:
} Select Select by barcode, and then scan the reagent plate barcode or enter the
reagent plate serial number.
} Select Select by name, and then select TruSeq Nano DNA.
5
Configure the run. Select Next.
} Select the Insert Size.
6
Review the run and sample information. Select Next.
7
Enter the consumable tracking information. Select Next.
8
Place the library card on the library card stage.
WARNING
To avoid instrument damage, make sure that the library card guide is not on the
library card.
9
Close the library card compartment door. Select Verify Library Card.
10 Place the library card guide on the library card.
11 Load the entire contents of the oil vial into the library card using the oil funnel.
WARNING
Use the required pipette tips. Other tips are not supported and can result in reagents not
dispensing properly and run failure.
The loading angle of the pipette depends on the item being dispensed. The angle is
specified in each step of the NPCS loading guide and is depicted in these procedures.
12 Transfer 45 µl of prepared samples 1–8.
13 Transfer 45 µl of prepared samples 9–16.
14 If you are preparing < 16 samples, add 45 µl RSB to empty sample wells.
15 Transfer 125 µl of the large reagents i–iv.
16 Transfer 125 µl of the large reagents v–vii.
17 Vortex DMB until well-dispersed.
18 Add 60 µl DMB to the large reagent well viii.
19 Transfer 15 µl of small reagents 1–4, and then 5–8.
20 Transfer 5 µl of small reagents a–d, and then e–h.
21 Transfer 3 µl of adapters A–H.
22 Transfer 3 µl of adapters I–P.
TruSeq Nano DNA Library Prep for NeoPrep Protocol Guide
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Set Up Run and Load Library Card
Set Up Run and Load Library Card
23 Remove the library card guide.
WARNING
To avoid instrument damage, make sure that the library card guide is removed from
the library card.
24 Close the library card compartment door. Select Start Run.
25 When the run is complete, select Next.
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Document # 15059579 v01
WARNING
The used library card contains hazardous materials. Personal injury can occur through
inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including
eye protection, gloves, and a laboratory coat. Handle the used library card as chemical
waste. Dispose of containers and any unused contents in accordance with the
governmental safety standards for your region. For more information, see the SDS for
this kit at support.illumina.com/sds.html.
Procedure
1
Add 10 µl RSB to each well of a new PCR plate labeled 1–16.
2
Open the library card compartment door and place the library card guide on the
library card.
3
Use a 200 µl pipette to transfer 20 µl from library card collection wells 1L–8L, and
then 9L–16L to corresponding wells 1–16 of the plate. Pipette to mix.
4
Centrifuge briefly.
5
Transfer the entire volume from plate wells 1–8, and then 9–16 to the center indent
in the membrane of the corresponding library separation tubes 1–16.
6
Let stand for 10 seconds while the oil is absorbed in the tubes.
7
Transfer the entire volume from library separation tubes 1–8, and then 9–16 to the
corresponding wells 1–16 of a new PCR plate.
8
Remove the library card and library card guide from the library card stage.
9
Discard the library card in accordance with applicable standards.
10 Close the library card compartment door, and then select Home.
11 Select from the following options:
Table 3 Post Run Options
NeoPrep
NeoPrep
System
System
Quantification Normalization
Yes
Yes
Pooling
Required
No
Yes
Yes
Yes
No
Yes
Yes or No
No
No
Yes or No
Then...
The protocol stops here. The final
library is normalized to 10 nM.
Proceed to cluster generation.
Proceed to Pool Libraries.
Proceed to
[Optional] Normalize Libraries
Manually.
Proceed to
[Optional] Validate Libraries
Manually.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.
TruSeq Nano DNA Library Prep for NeoPrep Protocol Guide
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Unload Libraries
Unload Libraries
[Optional] Validate Libraries Manually
Procedure
1
Quantify the libraries using a fluorometric quantification method that uses dsDNA
binding dyes or qPCR.
2
Check the library size distribution:
} If using a High Sensitivity DNA chip:
} Dilute the DNA library 1:10 with water.
} Run 1 µl diluted DNA library.
} If using a DNA 7500 chip, run 1 µl undiluted DNA library.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.
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Document # 15059579 v01
Procedure
1
Transfer 5 µl from each well of the library plate to the corresponding wells of a midi
plate.
2
Normalize each library to 10 nM with Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20.
Pipette to mix.
3
Select from the following options:
} For libraries that do not require pooling, the protocol stops here. Proceed to cluster
generation.
} For libraries that require pooling, proceed to Pool Libraries.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.
TruSeq Nano DNA Library Prep for NeoPrep Protocol Guide
9
[Optional] Normalize Libraries Manually
[Optional] Normalize Libraries Manually
Pool Libraries
Procedure
1
Determine the number of samples to combine for each pool.
2
Transfer 5 µl of each library to be pooled from the library plate to a single well of a
new PCR plate. Pipette to mix.
3
Proceed to cluster generation.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.
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Document # 15059579 v01
Acronyms
Acronyms
Acronym
Definition
DMB
Digital Microfluidics Beads
RSB
Resuspension Buffer
SC350
Sample Concentration Solution 350
SC550
Sample Concentration Solution 550
TruSeq Nano DNA Library Prep for NeoPrep Protocol Guide
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Notes
For technical assistance, contact Illumina Technical Support.
Table 4 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 5 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety data sheets (SDSs)—Available on the Illumina website at
support.illumina.com/sds.html.
Product documentation—Available for download in PDF from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
TruSeq Nano DNA Library Prep for NeoPrep Protocol Guide
Technical Assistance
Technical Assistance
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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