Tagment DNA

This snippet causes dropdowns to open on page load. This text/paragraph does not appear in the output.

This step uses Bead-Linked Transposomes for Enrichment to tagment double-stranded DNA. The tagmentation process fragments cDNA and adds adapter sequences.

After tagmentation, the fragments are purified and amplified to add index adapter sequences for dual indexing and P7 and P5 sequences for clustering. For help selecting index adapters, refer to Pooling Preparation.

• EBLTL (Bead-Linked Transposomes for Enrichment)
• EPM (Enhanced PCR Mix)
• ST2 (Stop Tagment Buffer 2)
• TB1 (Tagmentation Buffer 1)
• TWB (Tagmentation Wash Buffer)
• 1.7 ml microcentrifuge tube
• Index adapter plate
• Microseal 'B' adhesive film
• Nuclease-free ultrapure water
• UDP0XXX—Each well of the index adapter plate is single-use and contains > 10 µl UDP0XXX, which are premixed Index 1 (i7) and Index 2 (i5) adapters.
• The row and column labels are printed on the underside of the index adapter plate. Raise the plate overhead to check the labels.

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, refer to the SDS at support.illumina.com/sds.html.

1. Prepare the following consumables:
• EBLTL—Vortex to mix until beads are resuspended.
• EPM—Invert to mix, and then centrifuge briefly.
• Index adapter plate—Vortex to mix, and then centrifuge at 1000 × g for 1 minute.
• ST2—Vortex to mix, and then centrifuge briefly.
• TB1—Vortex to mix.
• TWB—Vortex to mix.
2. If the ST2 tube has precipitate, proceed as follows.
a. Heat at 37°C for 10 minutes.
b. Vortex until precipitate is dissolved.
c. Return to room temperature.
3. Save the following TAG program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 50 µl
• 55°C for 5 minutes
• Hold at 10°C

Total program time is ~5 minutes.

4. Save the following TAG_PCR program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 50 µl
• 72°C for 3 minutes
• 98°C for 3 minutes
• 17 cycles of:
• 98°C for 20 seconds
• 60°C for 30 seconds
• 72°C for 1 minute
• 72°C for 3 minutes
• Hold at 10°C

Total program time is ~50–60 minutes.

Tagment With EBLTL

1. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. In a 1.7 ml tube, combine the following volumes to prepare Tagmentation Master Mix. Multiply each volume by the number of samples.
• TB1 (11.5 µl)
• EBLTL (11.5 µl)
• Nuclease-free ultrapure water (14.5 µl)

Reagent overage is included in the volume.

3. Vortex the Tagmentation Master Mix to resuspend.
4. Add 32.5 µl Tagmentation Master Mix to each well.
5. Pipette to mix, and then seal.
6. Place on the preprogrammed thermal cycler and run the TAG program.

Each well contains a volume of 50 µl.

Wash Tagmented cDNA

1. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. Incubate at room temperature for 2 minutes.
3. Add 10 µl ST2 to each well.
4. Seal and shake at 2200 rpm for 1 minute.
5. Incubate at room temperature for 5 minutes.
6. Centrifuge at 280 × g for 10 seconds, and then unseal.
7. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
8. Remove and discard all supernatant.
9. Wash beads as follows.
a. Remove from the magnetic stand.
b. Add 100 µl TWB to each well.
c. Seal and shake at 2000 rpm for 1 minute.
d. Centrifuge at 280 × g for 3 seconds.
e. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
f. Remove and discard all supernatant.
10. Wash beads a second time.
11. Wash beads a third time. Do not discard supernatant.

TWB remains in the wells to prevent over-drying.

12. Keep on the magnetic stand.

Each well contains 100 µl beads with tagmented cDNA.

Amplify and Index Tagmented DNA

1. Combine the following volumes to prepare PCR Master Mix. Multiply each volume by the number of samples, and add the total calculated volumes to the 1.7 ml tube.
• EPM (23 µl)
• Nuclease-free ultrapure water (23 µl)

Reagent overage is included in the volume.

2. Vortex PCR Master Mix to mix.
3. Keeping the plate on the magnetic stand, remove and discard all TWB supernatant.
4. With a 20 µl pipette, remove all residual TWB.

Foam is normal and does not affect the library.

5. Remove from the magnetic stand.
6. Add 40 µl PCR Master Mix to each well.
7. Using a new pipette tip for each well, pierce the foil covering the index adapter plate wells that you intend to use.
8. Add 10 µl UDP0XXX to each well. Transfer volumes from the index adapter plate to the PCR plate.
9. Seal and shake at 2000 rpm for 1 minute.
10. Centrifuge at 280 × g for 3 seconds.
11. Place on the preprogrammed thermal cycler and run the TAG_PCR program.

Each well contains 50 µl beads with DNA attached.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at ‑25°C to ‑15°C for up to 7 days. Alternatively, leave on the thermal cycler for up to 24 hours.