1. No category

illumina TruSeq DNA Sample Prep. (LT) Protocol
1
Part# 15005180 Revision C June 2011
Performed using the TruSeq DNA Sample Preparation Kit (A cat#FC-121-1001, B cat#FC-121-1002)
Fragment DNA
Fragmentation will be conducted using the Covaris S2 Ultrasonicator
NOTE: Thaw Resuspension Buffer (10mM Tris pH 8.5) at RT˚
NOTE: Turn on the Covaris system >30 minutes before starting protocol. De-gas and pre-chill the deionized H2O to a temperature of
3˚C to 6˚C. The fragmentation procedure may be started when 6˚C is reached.
1. Dilute 1ug of gDNA to 55ul total volume in Resuspension Buffer for a final concentration of 20ng/ul in a 0.3ml PCR plate with a
DNA barcode label.
2. Transfer 52.5ul of diluted DNA from the DNA labeled plate to a Covaris tube. ADD VOLUME SLOWLY AVOIDING BUBBLES
3. Fragment the DNA using the following settings:
NOTE: For the Whole Genome Resequencing create the following program - 350bp DNA TruSeq micro_82211
Duty Cycle
Intensity
Bursts per second
Duration
Mode
Power
Temperature
Whole-genome Resequencing
10%
5.0
200
40 seconds
Frequency Sweeping
23W
5.5˚C – 6.0˚C
TruSeq Exome Enrichment
10%
5.0
200
120 seconds
Frequency Sweeping
23W
5.5˚C – 6.0˚C
NOTE: Make sure Covaris tube is properly positioned with the correct water level before initiating sonication.
4. Seal the Covaris tube and centrifuge at 600xg for 1 minute using an appropriate adaptor for the microcentrifuge rotor.
5. Transfer 50ul of the fragmented DNA from each Covaris tube to the corresponding well of a new 0.3ml PCR plate labeled with the
IMP barcode.
End Repair
NOTE: Thaw the following at RT˚: End Repair Control, End Repair Mix and Resuspension Buffer.
Mix well and centrifuge briefly. Place on ice.
NOTE: Remove the AMPure XP beads from 4˚C and let warm to RT˚ 30 minutes.
NOTE: Pre-heat thermal cycler to 30˚C.
NOTE: Apply an ALP barcode to a new 0.3ml PCR plate.
1. Add 10ul of the End Repair Control to each sample in the IMP plate containing 50ul of fragmented DNA. Substitute 10ul
Resuspension Buffer if End Repair Control is not used.
2. Add 40ul of End Repair Mix to each well of the IMP plate. Adjust volume of pipette to 100ul and pipette up and down 10x to mix.
3. Seal the IMP plate with a Microseal ‘B’ adhesive seal.
4. Incubate the IMP plate in a pre-heated thermal cycler at 30˚C for 30 min. using the following profile: 30HOLD
30˚C
hold
2
NOTE: Use heated lid
5. Remove IMP plate from thermal cycler promptly at 30 minutes.
IMP Clean Up
NOTE: Review AMPure XP bead handling on page 10 of the TruSeq DNA Sample Preparation Guide.
1. Vortex the pre-warmed AMPure XP Beads until they are well dispersed.
2. Do one of the following:
A. Gel Method
- Add 160ul of AMPure XP beads to each well of the IMP plate containing 100ul of End Repaired Mix.
B. Gel-Free Method
- Dilute AMPure XP beads by combining 125ul of well mixed beads with 35ul of nuclease free H 2O.
- Add 160ul of diluted bead mixture to each well of the IMP plate containing 100ul of End Repaired Mix.
3. Adjust pipette to 200ul and pipette up and down 10x to mix.
4. Incubate the IMP plate at RT˚ 15 minutes.
5. Transfer IMP plate to magnetic stand and incubate at RT˚ 15 min. or until liquid clears.
6. Remove 127.5ul of supernatant from each well of the IMP plate and discard. Change tip after each sample.
7. Remove a second 127.5ul volume from each well of the IMP plate. Change tip after each sample.
NOTE: Leave IMP plate on the magnetic stand while performing the following 80% ETOH washes.
NOTE: Prepare fresh 80% ETOH for all subsequent washing procedures.
8. Add 200ul 80% ETOH to each well of the IMP plate without disturbing the bead pellet.
9. Incubate the IMP plate at RT˚ for 30 seconds then remove and discard all supernatant from each well. DO NOT DISTURB
PELLET.
10. Repeat ETOH wash for a total of two 80% ETOH washes.
11. Let IMP plate stand at RT˚ for 15 min to dry then remove plate from magnetic stand.
12. Resuspend the dried pellet in each well with 17.5ul of Resuspension Buffer. Gently pipette entire volume up and down 10x to
mix thoroughly.
13. Incubate the IMP plate at RT˚ for 2 minutes.
14. Place the IMP plate on the magnetic stand at RT˚ for 5 minutes.
16. Transfer 15 ul of the clear supernatant from each well of the IMP plate to the corresponding well of a new 0.3ml PCR plate with
an ALP barcode.
NOTE: SAFE STOPPING POINT – seal the ALP plate with a Microseal ‘B’ adhesive seal and store at -25˚C for 7 days.
Adenylate 3’ Ends
NOTE: Thaw the following at RT˚: A-Tailing Control, A-Tailing Mix, Resuspension Buffer (if not using the A-Tailing Control).
NOTE: Thaw ALP plate at RT˚ if necessary and centrifuge briefly at 280xg for 1 minute. Remove adhesive seal.
NOTE: Pre-heat thermal cycler to 37˚C.
1. Add 2.5ul of the A-Tailing Control to each well of the ALP plate. Substitute 2.5ul Resuspension Buffer if A-Tailing Control is not
used.
2. Add 12.5ul of A-Tailing Mix to each well of the ALP plate.
3. Adjust the pipette to 30ul and gently pipette the entire volume up and down 10x to mix. Change tip after each sample.
4. Incubate the ALP plate in a pre-heated thermal cycler at 37˚C for 30 minutes using the following profile: 37HOLD
37˚C
hold
NOTE: Use heated lid
5. Immediately remove the ALP from the thermal cycler then proceed immediately to Ligate Adapters.
Ligate Adapters
NOTE: Thaw the following at RT˚: Appropriate DNA Adapter Index tubes (AD001-AD012), Stop Ligation Buffer, Ligase Control,
Resuspension Buffer .
NOTE: Remove the AMPure XP beads from 4˚C and let warm to RT˚ 30 minutes.
NOTE: Pre-heat thermal cycler to 30˚C.
NOTE: Apply a CAP barcode label to a new 0.3ml PCR plate.
NOTE: Apply a SSP barcode label to a new 0.3ml PCR plate if using the gel method.
Apply a PCR barcode label to a new 0.ml PCR plate if using the gel-free method.
1. Mix well and briefly centrifuge the thawed DNA Adapter Index tubes, Ligase Control and Stop Ligation Buffer tubes at 600xg.
2. Remove the adhesive seal from the ALP plate.
3. Add 2.5ul of the Ligase Control to each well of the ALP plate. Substitute 2.5ul Resuspension Buffer if A-Tailing Control is not
used.
4. Immediately before use, remove the Ligation Mix from -25˚C storage and place on ice.
5. Add 2.5ul of Ligation Mix to each well of the ALP plate
6. Immediately return the Ligation Mix back to -25˚C storage after use.
7. Add 2.5ul of the appropriate DNA Adapter Index to each well of the ALP plate.
8. Adjust the pipette to 37.5ul and pipette the entire volume up and down 10x to mix.
9. Seal the ALP plate with a Microseal ‘B’ adhesive seal.
10. Incubate the ALP plate in a pre-heated thermal cycler at 30˚C for 10 minutes using the following profile: 30HOLD
30˚C
hold
NOTE: Use heated lid
11. Remove the ALP plate from the thermal cycler and remove seal.
12 Add 5ul of Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation.
13. Adjust the pipette to 42.5ul and gently pipette up and down 10x to mix thoroughly.
ALP Clean Up
1. Vortex the AMPure XP beads until they are well dispersed.
2. Add 42.5ul of beads to each well of the ALP plate. NOTE: Keep beads at RT˚ for use later in this clean-up procedure.
3. Adjust the pipette to 85ul and pipette the entire volume up and down 10x to mix thoroughly
4. Incubate the ALP plate at RT˚ for 15 minutes.
5. Transfer the ALP plate to the magnetic stand at RT˚ for 5 minutes.
6. Remove and discard 80ul of the supernatant from each well.
NOTE: Leave IMP plate on the magnetic stand while performing the following 80% ETOH washes.
3
4
7. With the ALP remaining on the magnetic stand, add 200ul of freshly prepared 80% ETOH to each well. DO NOT DISTURB
PELLET.
8. Incubate the ALP plate at RT˚ for 30 seconds. Remove and discard the supernatant from each well avoiding pellet.
9. Repeat wash steps for a total of two 80% ETOH washes.
10. Keep the ALP plate on the magnetic stand and allow to air dry at RT˚ for 15 minutes. Remove ALP plate from magnetic stand.
11. Resuspend the dried pellet with 52.5ul of Resuspension Buffer. Gently pipette up and down 10x to mix thoroughly
12. Incubate the RLP plate at RT˚ 2 minutes.
13 Place the ALP plate on the magnetic stand at RT˚ for 5 minutes.
14. Transfer 50ul of cleared supernatant from the ALP plate to the corresponding well of the new 0.3ml plate with a CAP barcode.
15. Add 50ul of freshly vortexed AMPure XP beads to each well of the CAP plate for a second clean-up.
16. Adjust the pipette to 100ul and gently pipette up and down 10x to mix thoroughly.
17. Incubate the CAP plate at RT˚ for 15 minutes.
18. Place the CAP plate on the magnetic stand for 5 minutes.
19. Remove and discard 95ul of the supernatant from each well of the CAP plate.
NOTE: Leave IMP plate on the magnetic stand while performing the following 80% ETOH washes.
20. With the CAP plate remaining on the magnetic stand, add 200ul freshly prepared 80% ETOH to each well. DO NOT DISTURB
PELLET.
21. Incubate the CAP plate at RT˚ 30 seconds then remove and discard all the supernatant without disturbing the pellet.
22. Repeat wash steps for a total of two 80% ETOH washes.
23. Keep the CAP plate on the magnetic stand and air dry the pellets at RT˚ for 15 minutes. Remove plate from magnetic stand.
24. Resuspend the dried pellet in 22.5ul of Resuspension Buffer and gently pipette up and down 10x to mix thoroughly.
25. Incubate the CAP plate at RT˚ for 2 minutes.
26. Place the CAP plate on the magnetic stand for 5 minutes at RT˚.
27. Do one of the following:
A. Gel Method
- Transfer 20ul of the clear supernatant from the CAP plate to the corresponding well of 0.3ml SSP plate.
- Proceed to Purify Ligation Products (gel method only) section
B. Gel-Free Method
- Transfer 20ul of the clear supernatant from the CAP plate to the corresponding well of 0.3ml PCR plate.
- Proceed to Enrich DNA Fragments section.
NOTE: SAFE STOPPING POINT – seal the SSP plate or PCR plate with a Microseal ‘B’ adhesive seal and store at -25˚C
for 7 days.
Purify Ligation Products (Gel Method Only)
Perform the size selection using the Sage Pippin Prep NOTE: Bring Pippin Prep loading solution to RT˚.
Size Selection options:
Whole Genome Resequencing
TruSeq Exome Enrichment
A
Insert Size Target
300-400bp
200-300bpA
Gel Slice location
400-500bpB
300-400bpB
A -Targeted insert size without adapters. B – Cut size adjusted for adaptor sequence.
Prepare DNA Samples for loading
1. Initiate the Pippin Prep instrumentation allowing software to launch.
2. Add 10ul of Resuspension Buffer to each sample in the SSP plate to obtain 30ul total volume.
3. Add 10ul of RT˚ loading solution to each sample for a total of 40ul. Mix well and centrifuge briefly.
Program Protocol
5
1. From main screen select “Protocol” to go to the Protocol Editor Screen. Press “New” to open a new protocol.
2. Select the appropriate cassette type ( 2% Marker B No Overflow Detection).
3. Select a reference lane which will contain the DNA marker. NOTE: Verify lane assignment of software display vs cassette lanes.
4. Enter - bp target size 450bp
-bp start size 400bp
-bp end size 500bp
5. Enter sample name into the “Sample ID” field.
6. Select “Save as” and name protocol to retain for future applications (TruSeq DNA 400-500bp).
7. Press “Return” to return to Run Screen.
Prepare Cassette
1. Remove cassette from packaging and wipe off excess moisture. Verify correct cassette type is used.
2. Visually inspect buffer chamber and gel columns. From side view ensure buffer chamber volume is more than ½ full. Inspect gel
column for breakage, bubbles or gaps. Any lanes with these defects must be abandoned. Remaining lanes are still functional.
3. Inspect for air gaps in and behind elution wells by tilting cassette to the left (loading well end). Tap on bench lightly to dislodge.
4. Install cassette into tray of Pippin Prep with loading wells to the left.
5. Remove sealing tape without tipping the cassette.
6. Remove all buffer from elution well and replace with 40ul of electrophoresis buffer.
7. Test electrophoretic current continuity:
a. Close sliding lid on instrument.
b. In the run screen select “Manual Mode” from the “Protocol Name” drop down menu.
c. Press “Test” in controller on the Main Screen. Test takes 30 sec. finishing with “Pass” of “Fail” message.
d. If test fails – in the separation lane: lane unusable – no recovery
- in the elution lane: Verify 40ul of running buffer is present in the elution well and re-test. If it fails again lane
can be used for reference sample.
7. Cover all collection wells with tape to minimize recover volume to ~50ul.
Sample Loading
1. Verify that sample wells are completely full. Top off wells with electrophoresis buffer if necessary. Total well volume is 70ul.
2. Remove 40ul of running buffer from the loading well leaving ~30ul in well.
3. Load 40ul of pre-mixed Pippin Prep DNA Marker into the sample lane that has been assigned for the reference. (Reference lane can
be changed as needed).
4. Load 40ul of the supplemented sample into the appropriate lane loading well.
5. Close lid
6. Select desired protocol from the “Protocol Name” drop down menu. (ie: TruSeq DNA 400-500)
7. Press “Start”.
Sample Collection
1. When complete remove elution well tape and transfer all sample from sample elution well to a labeled microcentrifuge tube.
2. Determine volume of collected sample. Volume can be from 40-70ul.
3. Clean electrodes of Pippin Prep by installing cleaning cassette filled with H2O and closing lid for 30 seconds. Remove Cassette.
Sample Clean-Up
Use the Qiagen MinElute PCR Purification Kit (cat# 28004) for clean-up procedure. Perform the PCR Purification
Protocol.
NOTE: Must add 120ul of the pH Indicator to 30ml of the PB buffer before proceeding.
: Add ETOH to the wash buffer PE
1. Add 5 volumes of PB buffer (with Indicator) to 1 volume of the Pippin Prep elute (250ul PB+I buffer + 50ul Pippin Prep Elute).
2. Check the color of the mixture and ensure that it is yellow (similar to the original color of buffer ERC).
a. If color is orange or violet add 10ul of 3M sodium acetate pH 5.0 and mix. The color will return to yellow.
6
3. Place a MinElute column in a 2ml collection tube.
4. Bind DNA by adding the sample to the MinElute column and centrifuging 1 min at 13,000 RPM at RT˚
5. Discard the flow through and place the MinElute column back in the same collection tube.
6. Wash column by adding 750ul of Buffer PE to the MinElute column and centrifuge 1 min at 13,000 RPM at RT˚
7. Discard flow-through and place MinElute column back in the same collection tube.
8. Centrifuge MinElute column for an additional 1 minute to remove any residual wash buffer PE.
9. Place the MinElute Column in a new labeled 1.5ml microcentrifuge tube.
10. Elute DNA by adding 25ul of EB buffer (10mM Tris-HCl pH 8.5) to the center of the membrane. Let column stand at RT˚ 1
minute.
11. Centrifuge MinElute column for 1 minute at 13,000 RPM.
12. Transfer 20ul of purified sample to the appropriate well of a 0.3ml plate labeled with a PCR barcode.
NOTE: SAFE STOPPING POINT – seal the PCR plate with a Microseal ‘B’ adhesive seal and store at -25˚C
for 7 days before proceeding to Enrich DNA Fragments.
Enrich DNA Fragments
NOTE: Thaw the following at RT˚: PCR Master Mix and PCR Primer Cocktail. Spin briefly at 600xg and transfer to ice.
: Resuspension Buffer
NOTE: Remove the AMPure XP beads from 4˚C and let warm to RT˚ 30 minutes.
NOTE: Preheat thermal cycler by initiating the DNAAMP profile.
NOTE: Apply a TSP1 barcode label to a new 0.3ml PCR plate.
Make PCR (PCR procedure assumes 1ug of input DNA to the library prep to obtain high yield libraries)
1. Add 5ul of PCR Primer Cocktail to the 20ul sample in the PCR plate.
2. Add 25ul of PCR Master Mix to each sample in the PCR plate yielding 50ul total volume.
3. Adjust the pipette to 40ul and pipette up and down 10x to mix thoroughly.
4. Seal the plate with a Microseal ‘A’ film according to manufacturer’s instructions.
NOTE: Using Microsael’B’ adhesive seal for PCR may cause droplets of sample to splash onto the film when peeling the
film from the plate.
5. Transfer PCR plate to a pre-warmed thermal cycle and amplify using the following profile: DNAAMP
1 cycle
98˚C 30 sec
10 cycles
98˚C 10 sec
60˚C 30 sec
72˚C 30 sec
1 cycle
72˚C 5 min
4˚C
Hold
NOTE: use heated lid
6. Remove adhesive ‘A’ seal from the plate.
7. Vortex the pre-warmed RT˚ AMPure XP beads until they are well dispersed.
7
8. Add 50ul of AMPure XP beads to each sample in the PCR plate for a 100ul total volume. Pipette up and down 10x to mix well.
9. Incubate the PCR plate at RT˚ for 15 minutes.
10. Place the PCR plate on the magnetic stand at RT˚ for 5 minutes.
11. With the PCR plate on the magnetic stand, remove and discard 95ul of the supernatant from each well of the PCR plate.
NOTE: Leave PCR plate on the magnetic stand while performing the following 80% ETOH washes.
12. Keeping the PCR plate on the magnetic stand, add 200ul of freshly prepared 80% ETOH to each well. DO NOT DISTURB
PELLET.
13. Incubate the PCR plate at RT˚ for 30 seconds then remove and discard all of the supernatant from each well.
14. Repeat ETOH wash step one more time for a total of two 80% ETOH washes.
15. Keeping the PCR plate on the magnetic stand allow the plate to air dry at RT˚ for 15 minutes.
16. Remove PCR plate from magnetic stand and resuspend each dried pellet with 32.5ul of Resuspension Buffer. Gently pipette up
and down 10x to mix thoroughly.
17. Incubate the plate at RT˚ for 2 minutes.
18. Place the PCR plate on the magnetic stand at RT˚ for 5 minutes.
19. Transfer 30ul of the cleared supernatant to the corresponding well of a new 0.3ml plate labeled with a TSP1 barcode.
20. Do one of the following:
A. If performing Whole-Genome Resequencing, proceed to Validate Library.
B. If performing TruSeq Enrichment, proceed to the TruSeq Enrichment Guide for instructions on how to quantify and
qualify your library.
NOTE: SAFE STOPPING POINT – If you do not plan to proceed to Validate Library or TruSeq Enrichment,
seal the PCR plate with a Microseal ‘B’ adhesive seal and store at -25˚C
for up to 7 days before proceeding.
Validate Library
1. Determine the concentration of each amplified library using the Nanodrop.
2. Perform QC of the amplified library by making a 1:50 dilution of each library in H 2O and loading 1ul of the diluted library on the
Agilent High Sensitivity DNA chip.
NOTE: Agilent run is for qualitative purposes only. A single peak should be present at ~450bp.
3. Calculate the nM concentration of each library utilizing the Ambion RNA and DNA Concentration Calculator available at
http://www.ambion.com/techlib/append/concentration_calculator.html
4. Transfer 10ul of each library from the TSP1 plate to a new Midi plate labeled with a DCT barcode and dilute to 10nM concentration
using EB buffer (10mM Tris HCL pH 8.5) supplemented with 0.1% Tween20.
5. Pipet up and down with multichannel pipette 10x to mix.
6. For non-multiplexed samples that do not require pooling proceed to cluster generation.
NOTE: Store DCT barcoded MIDI plate with diluted libraries at -20˚C until ready to generate clusters.
NOTE: Libraries diluted to 10nM in EB buffer supplemented 0.1% Tween20 are safe for long term storage at -20˚C
Pooling Libraries (Optional)
1. Apply a PDP barcode label to a new 0.3ml plate (for multiplexing only).
2. Determine the samples to be combined together for each pool.
NOTE: Verify that libraries to be pooled have unique indexes!!
3. Transfer 10 ul of each normalized library to be pooled from the DCT plate to one well of the new PDP labeled plate. The total
volume of the combines sample libraries will 10x the number of combined samples (6 libraries = 60ul).
4. Pipette the entire volume up and down 10x to thoroughly mix the pooled libraries.
5. Proceed to cluster generation.
8