TruSeq Nano DNA Library Prep Checklist (15075698 A)

TruSeq Nano DNA Library Prep Checklist (15075698 A)
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Nano DNA Library Prep
Fragment DNA
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□6
Normalize gDNA with RSB to 52.5 µl in the
DNA plate.
• 100 ng for a 350 bp insert size
• 200 ng for a 550 bp insert size
Mix thoroughly.
Centrifuge.
Transfer 52.5 µl DNA to Covaris tubes.
Centrifuge at 280 × g for 5 seconds.
Fragment the DNA using the following settings.
□12 Place on a magnetic stand until liquid is clear.
□13 Remove and discard all supernatant.
□14 Wash 2 times with 200 µl 80% EtOH.
□15 Use a 20 µl pipette to remove residual EtOH.
□16 Air-dry for 5 minutes.
□17 Add 62.5 µl RSB.
□18 Remove from the magnetic stand and mix.
□19 Incubate at room temperature for 2 minutes.
□20 [HS] Centrifuge at 280 × g for 1 minute.
□21 Place on a magnetic stand until liquid is clear.
□22 Transfer 60 µl supernatant to the IMP plate.
Table 1 350 bp Insert Settings
M220 S220
S2
E210
Setting
Duty Factor (%)
20
5
10
Intensity
—
—
5.0
Power (W)
50
175
23
14
Cycles/Burst
200
Duration (sec)
65
50
45
Mode
—
Frequency sweeping
Temperature (°C)
20
5.5–6
Table 2 550 bp Insert Settings
M220 S220
S2
E210
Setting
Duty Factor (%)
20
5
10
Intensity
—
—
2.0
Power (W)
50
175
9
7
Cycles/Burst
200
Duration (sec)
45
25
45
Mode
—
Frequency sweeping
Temperature (°C)
20
5.5–6
□1
□2
□3
□4
□5
□6
□7
□8
□9
□10
□11
□12
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□14
□15
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□17
□18
□19
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□21
□22
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Add 40 µl ERP2/ERP3 and mix.
[HS] Centrifuge at 280 × g for 1 minute.
Incubate as follows.
• [HS] Place on the 30°C microheating system for
30 minutes, and then place on ice.
• [LS] Place on the thermal cycler and run the
ERP program.
Dilute SPB with PCR grade water to 160 µl per
100 µl of sample.
Vortex diluted SPB until well-dispersed.
Add 160 µl diluted SPB and mix.
Incubate at room temperature for 5 minutes.
[HS] Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand until liquid is clear.
Transfer 250 µl supernatant to the CEP plate.
Add 30 µl SPB and mix.
Incubate at room temperature for 5 minutes.
[HS] Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Wash 2 times with 200 µl 80% EtOH.
Use a 20 µl pipette to remove residual EtOH.
Air-dry for 5 minutes.
Add 20 µl RSB.
Remove from the magnetic stand and mix.
Incubate at room temperature for 2 minutes.
[HS] Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand until liquid is clear.
Transfer 17.5 µl supernatant to the ALP plate.
SAFE STOPPING POINT
□7 Centrifuge at 280 × g for 5 seconds.
□8 Transfer 50 µl supernatant to the CSP plate.
□9 Add 80 µl SPB and mix.
□10 Incubate at room temperature for 5 minutes.
□11 [HS] Centrifuge at 280 × g for 1 minute.
Part # 15075698 Rev. A
Repair Ends and Select Library Size
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 7 days.
June 2015
ILLUMINA PROPRIETARY
Page 1 of 4
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Nano DNA Library Prep
Adenylate 3ʹ Ends
□1
□2
□3
Add 12.5 µl ATL/ATL2 and mix.
Centrifuge at 280 × g for 1 minute.
Incubate as follows.
[HS]
a Place on the 37°C microheating system for
30 minutes.
b Move to the 70°C microheating system for
5 minutes.
c Place on ice for 5 minutes.
[LS]
a Place on the thermal cycler and run the
ATAIL70 program.
b Centrifuge at 280 × g for 1 minute.
□1
Add the following and mix.
Reagent
RSB
LIG2
DNA adapters
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□
□
□2
□3
□
□
□7
□8
Ligate Adapters
□4
□5
□6
Volume (µl)
2.5
2.5
2.5
□9
Transfer 50 µl supernatant to the CAP plate.
Repeat steps 6a through 6m with the new plate
using the Round 2 volumes.
Transfer 25 µl supernatant to the 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.
Centrifuge at 280 × g for 1 minute.
Incubate as follows.
• [HS] Place on the 30°C microheating system for
10 minutes, and then place on ice.
• [LS] Place on the thermal cycler and run the
LIG program.
Add 5 µl STL and mix.
[HS] Centrifuge at 280 × g for 1 minute.
Perform steps 6a through 6m using the Round 1
volumes.
a Add SPB and mix.
□
SPB
Round 1
42.5 µl
Round 2
50 µl
□b Incubate at room temperature for 5 minutes.
□c [HS] Centrifuge at 280 × g for 1 minute.
□d Place on a magnetic stand until liquid is clear.
□e Remove and discard all supernatant.
□f Wash 2 times with 200 µl 80% EtOH.
□g Use a 20 µl pipette to remove residual EtOH.
□h Air-dry for 5 minutes.
□i Add RSB.
RSB
Round 1
52.5 µl
Round 2
27.5 µl
□j Remove from the magnetic stand and mix.
□k Incubate at room temperature for 2 minutes.
□l [HS] Centrifuge at 280 × g for 1 minute.
□mPlace on a magnetic stand until liquid is clear.
Page 2 of 4
June 2015
ILLUMINA PROPRIETARY
Part # 15075698 Rev. A
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Nano DNA Library Prep
Enrich DNA Fragments
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Place on ice and add 5 µl PPC.
Add 20 µl EPM and mix.
Centrifuge at 280 × g for 1 minute.
Place on the thermal cycler and run the
PCRNano program.
Centrifuge at 280 × g for 1 minute.
Add SPB.
Adapter Type
Adapter tubes
DAP
Validate Libraries
□1
□2
Quantify the libraries.
Do the following:
• If using a High Sensitivity DNA chip:
— Dilute the DNA library 1:100 with water.
— Run 1 µl diluted DNA library.
• If using a DNA 7500 chip, run 1 µl undiluted
DNA library.
Normalize and Pool Libraries
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□2
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□5
Volume SPB
50 µl
47.5 µl
□6
□7 Mix thoroughly.
□8 Incubate at room temperature for 5 minutes.
□9 [HS] Centrifuge at 280 × g for 1 minute.
□10 Place on a magnetic stand until liquid is clear.
□11 Remove and discard all supernatant.
□12 Wash 2 times with 200 µl 80% EtOH.
□13 Use a 20 µl pipette to remove residual EtOH.
□14 Air-dry for 5 minutes.
□15 Add 32.5 µl RSB.
□16 Remove from the magnetic stand and mix.
□17 Incubate at room temperature for 2 minutes.
□18 [HS] Centrifuge at 280 × g for 1 minute.
□19 Place on a magnetic stand until liquid is clear.
□20 Transfer 30 µl supernatant to the TSP1 plate.
Transfer 10 µl library to the DCT plate.
Normalize with Tris-HCl 10 mM, pH 8.5 with
0.1% Tween 20 to 10 nM and mix.
[HS] Centrifuge at 280 × g for 1 minute.
If pooling 2–24 samples, transfer 10 µl to a single
well of the PDP plate.
If pooling 25–48 samples.
a Transfer 5 µl to column 1 of the PDP plate and
mix.
b [HS] Centrifuge at 280 × g for 1 minute.
c Transfer column 1 to well A2.
If pooling 49–96 samples.
a Transfer 5 µl to column 1 of the PDP plate and
mix.
b [HS] Centrifuge at 280 × g for 1 minute.
c Transfer column 1 to a 1.7 ml microcentrifuge
tube.
Mix thoroughly.
[HS] Centrifuge at 280 × g for 1 minute.
Proceed to cluster generation.
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□
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□
□7
□8
□9
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube
and store at -25°C to -15°C for up to 7 days.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 7 days.
Part # 15075698 Rev. A
June 2015
ILLUMINA PROPRIETARY
Page 3 of 4
TruSeq Nano DNA Library Prep
For Research Use Only. Not for
use in diagnostic procedures.
Acronyms
Acronym
Definition
ALP
Adapter Ligation Plate
ATL
A-Tailing Mix
CAP
Clean Up ALP Plate
CEP
Clean Up End Repair Plate
CSP
Clean Up Sheared DNA Plate
DAP
DNA Adapter Plate
DCT
Diluted Cluster Template Plate
DNA
Customer Sample DNA Plate
EPM
Enhanced PCR Mix
ERP
End Repair Mix
IMP
Insert Modification Plate
LIG
Ligation Mix
PDP
Pooled Dilution Plate
PPC
PCR Primer Cocktail
RSB
Resuspension Buffer
SPB
Sample Purification Beads
STL
Stop Ligation Buffer
TSP1
Target Sample Plate 1
Page 4 of 4
June 2015
ILLUMINA PROPRIETARY
Part # 15075698 Rev. A
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