Bead-Based Normalization

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This step uses bead-based normalization to normalize the quantity of each library to obtain a uniform library representation in the sequencing pool.

• EE1 (Enrichment Elution Buffer 1)
• HP3 (2 N NaOH)
• LNA1 (Library Normalization Additives)
• LNB1 (Library Normalization Beads)
• LNS1 (Library Normalization Storage Buffer 1)
• LNW1 (Library Normalization Wash 1)
• RSB (Resuspension Buffer)
• 1.7 ml microcentrifuge tube
• 96-well storage plate, deep-well
• 96-well PCR plate, LoBind
• Microseal 'B' adhesive seals

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.

Aspirate and dispense LNB1 beads slowly due to the viscosity of the solution.

1. Prepare the following consumables:
• LNA1—Vortex vigorously to make sure that all precipitates have dissolved.
• LNB1:
a. Pulse vortex on highest setting for 1 minute.
b. Pipette to mix with a 1000 μl pipette set to 800 μl.
c. Repeat steps a and b until all beads are resuspended. Visually confirm that no beads are present at the bottom of the tube when it is inverted.

It is critical to completely resuspend the bead pellet at the bottom of the tube. Resuspension is essential to achieve consistent cluster density.

• LNS1—vortex to mix.
• LNW1—vortex to mix.
• HP3—vortex to mix, and then centrifuge briefly.
• EE1—vortex to mix, and then centrifuge briefly.
2. Combine the following volumes to prepare LNA1 + LNB1 Master Mix. Multiply each volume by the number of samples.
• LNA1 (47.6 μl)
• LNB1 (8.6 μl)

These volumes produce 56.2 μl LNA1 + LNB1 Master Mix per sample, which includes extra volume for accurate pipetting.

3. Vortex LNA1 + LNB1 Master Mix to mix.
4. Combine the following volumes to prepare EE1 + HP3 Master Mix. Multiply each volume by the number of samples.
• EE1 (38 μl)
• HP3 (2 μl)

These volumes produce 40 μl EE1 + HP3 Master Mix per sample, which includes extra volume for accurate pipetting.

5. Prepare the PL plate:
a. Centrifuge briefly.
b. Pipette to mix.
6. Label a new 96-well deep-well plate BBN.
7. Label a new 96-well LoBind PCR plate NL.

Bind

1. Vortex LNA1 + LNB1 Master Mix briefly.
2. Add 45 μl LNA1 + LNB1 Master Mix to each well of the BBN plate.
3. Add 20 μl from each well of the PL plate to the corresponding well of the BBN plate.
4. Seal and shake at 1800 rpm for 10 minutes.
5. Place on a peg magnetic stand and wait until the liquid is clear (2 minutes).
6. Without disturbing the beads, remove and discard all supernatant from each well.

Wash

1. Vortex LNW1 briefly.
2. Wash the beads as follows.
a. Remove the BBN plate from the magnetic stand and add 45 μl LNW1 to each well.
b. Seal and shake at 1800 rpm for 2 minutes.
c. Place on a peg magnetic stand and wait until the liquid is clear (2 minutes).
d. Without disturbing the beads, remove and discard all supernatant from each well.
3. Repeat step 2 to wash the beads a second time.
4. Use a 20 μl pipette to remove residual supernatant from each well.

Elute

1. Vortex EE1 + HP3 Master Mix briefly.
2. Remove the BBN plate from the magnetic stand and add 32 μl EE1 + HP3 Master Mix to each well.
3. Seal and shake at 1800 rpm for 2 minutes.
4. Place on a peg magnetic stand and wait until the liquid is clear (2 minutes).
5. Add 30 μl LNS1 to each well of the NL plate.
6. Without disturbing the beads, transfer 30 μl supernatant from each well of the BBN plate to the corresponding well of the NL plate. Pipette five times to mix.
7. Discard the BBN plate.

SAFE STOPPING POINT

If you are stopping, apply Microseal 'B' to the NL plate, and briefly centrifuge at 280 × g. Store at 2°C to 8°C overnight, or at ‑25°C to ‑15°C for up to 30 days.