Append Index Adapters

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This step attaches adapters required for sequencing cluster generation to each sample to create a library of short, overlapping fragments.

Index adapter plates are ordered separately. Refer to Product Contents for details.

Make sure that the indexes used are unique for each library. Some indexes in the Set B and Set D Illumina DNA/RNA Unique Dual Indexes (96 Indexes, 96 Samples) plates are also used in the Illumina DNA/RNA Unique Dual Indexes (48 Indexes, 48 Samples) plate. Use of non-unique indexes can lead to failure in sample differentiation and FASTQ generation.

• Enhanced PCR Mix 4 (EPM4)
• Resuspension Buffer (RSB)
• Stop Tagment Buffer 2 (ST2)
• Tagmentation Wash Buffer 2 (TWB2)
• Illumina DNA/RNA Unique Dual Indexes Plate (UDP)
• 1.7 ml microcentrifuge tube

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Ventilation should be appropriate for handling of hazardous materials in reagents. 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.

• ST2
– Pipette slowly to minimize foaming.
• TWB2
– Dispense TWB2 directly onto the beads.
– Pipette slowly to minimize foaming.
1. Prepare the following consumables:
• EPM4—Vortex to mix, and then centrifuge briefly.
• RSB—Vortex to mix.
• ST2—Vortex until precipitates are dissolved.
• TWB2—Invert to mix.
• Illumina DNA/RNA Unique Dual Indexes Plate (UDP)—Vortex to mix, and then centrifuge briefly.
2. Save the following INDEX PCR program on a thermal cycler:
• Choose the preheat lid option and set to 105°C
• Set the reaction volume to 50 µl
• 68°C for 3 minutes
• 98°C for 3 minutes
• 6 cycles of:
• 98°C for 45 seconds
• 62°C for 30 seconds
• 68°C for 2 minutes
• 68°C for 1 minute
• Hold at 4°C
1. Remove from the thermal cycler and add 10 μl ST2 to each well.
2. Using a pipette set to 30 µl, pipette to mix.
3. Incubate at room temperature for 2 minutes.
4. Wash beads as follows.
a. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
b. Remove and discard all supernatant from each well.
c. Remove from the magnetic stand and add 100 μl TWB2 to each well.
d. Pipette to mix until beads are fully resuspended.
5. Wash beads a second time.

After the second wash, keep pellets in TWB2 to prevent overdrying of the beads.

6. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
7. In a 1.7 ml tube, combine the following volumes to prepare Index Master Mix. Multiply each volume by the number of samples.
• RSB (28.6 µl)
• EPM4 (22 µl)

These volumes produce 50.6 µl Index Master Mix per sample, which includes overage.

8. Vortex to mix, and then centrifuge briefly.
9. Remove and discard all TWB2 from each well.
10. Using a 20 µl pipette, remove any residual TWB2 from each well without disturbing the bead pellet.
11. Remove from the magnetic stand and add 46 μl Index Master Mix to each well.
12. Pipette to mix until beads are fully resuspended.
13. Centrifuge UDP briefly.
14. Add 4 µl index adapters from UDP to each well.
15. Using a pipette set to 40 µl, pipette to mix.
16. Seal the plate, place on the preprogrammed thermal cycler, and run the INDEX PCR program.