Hybridize DNA to the BeadChip

This step dispenses the fragmented, resuspended DNA onto BeadChips. Incubation then hybridizes each DNA sample to a section of the BeadChip.

Before hybridizing DNA to the BeadChip make sure that the DMAP files have been downloaded for all BeadChips that will be used.

• 100% EtOH (Ethanol)
• PB2
• XC4
• Keep XC4 in the original bottle until you are ready to use it.
• Each XC4 bottle contains sufficient reagent to process up to 48 BeadChips.
• Use resuspended XC4 at room temperature.
1. If frozen, thaw the MSA4 plate at room temperature, and then pulse centrifuge at 280 × g.
2. Preheat the heat block to 95°C.
3. Preheat the Illumina Hybridization Oven to 48°C.

Procedure

1. Place the MSA4 plate at 95°C on the preheated heat block and incubate it for 20 minutes to denature the DNA.
2. Cool the MSA4 plate on the benchtop at room temperature for 30 minutes.
3. Pulse centrifuge at 280 × g.

Assemble one chamber for every four BeadChips by following the steps in this section.

1. Place the hybridization chambers, chamber gaskets, and chamber inserts on the benchtop.

Hybridization Chamber Components

A. Hybridization chambers
B. Hybridization chamber gaskets
C. Hybridization chamber inserts
2. Align the wider edge of the gasket to the barcode ridges.

Gasket and Hybridization Chamber Alignment Components

A. Reagent reservoirs
B. Barcode ridges
C. Narrower gasket edges
D. Wider gasket edges
3. Place the gasket into the hybridization chamber as follows.
a. Match the wider edge of the hybridization chamber gasket to the barcode-ridge side of the hybridization chamber.
b. Press down on the edges of the gasket to make sure it is properly seated.

Placing of Gaskets on Hybridization Chamber

4. Make sure that the gaskets are properly placed and seated.

Proper Gasket Placement on Hybridization Chamber

5. Add 400 µl PB2 into each of the eight humidifying buffer reservoirs in the hybridization chamber.

PB2 Addition

6. Immediately cover the chamber with the lid to prevent evaporation.
7. Leave the closed chambers on the benchtop at room temperature until the BeadChips are loaded with DNA (~1 hour).
8. [Illumina LIMS] In Illumina LIMS, select HD Methylation Assay | Confirm for Hyb.
a. Scan the barcode of each MSA4 plate you plan to hybridize.
b. Scan the BeadChip barcode on the package of each BeadChip you plan to hybridize.
c. Select Verify.
1. Remove the BeadChips from packaging. Hold BeadChips by the ends, away from the sample inlets.
2. Place each BeadChip into an insert so that the barcode ends align.

BeadChip Placement in Insert

3. Transfer the appropriate volume of each sample from the MSA4 plate to the appropriate section of the BeadChip.

Make sure that the pipette tip is in the sample inlet before dispensing.

• 8x1 BeadChips: 26 µl each sample
• 12x1 BeadChips: 15 µl each sample
• Load A1–H1, as shown in the following graphic.
• Repeat for each column until all samples are loaded.

MSA4 Plate Layout for 8x1 BeadChip

MSA4 Plate Layout for 12x1 BeadChip

4. Wait for the DNA to disperse over the entire surface.
5. Inspect the loading port for excess liquid.

Example Excess Liquid

6. If excess liquid is not present, add leftover sample from the amplification plate to create a bolus around the loading port. Do not use RA1, which dilutes the sample.

Excess liquid prevents evaporation and the creation of low-intensity areas.

7. Store RA1 at -25°C to -15°C for use the next day.
8. Heat-seal any residual sample in the MSA4 plate with foil.

SAFE STOPPING POINT

If you are stopping, seal the plate, and store at -80°C indefinitely.

[Optional] If you are not stopping, you can set aside the MSA4 plate for up to 1 hour before proceeding.

To ensure accurate results, record and track each BeadChip and the hybridization chamber it is added to.

1. Load the inserts containing BeadChips into the hybridization chamber.
• Position the barcode end over the ridges indicated on the chamber.
• Keep the inserts steady and level.

Loading of Inserts into Chamber

2. Place the back of the lid onto the chamber, and then slowly lower the front to avoid dislodging the inserts.

Lowering of Chamber Lid

3. Close all four clamps so that the lid is secure and sits evenly on the base without any gaps.

Close the clamps in the following order: top-left, bottom-right, top-right, bottom-left.

4. Place the chamber into the preheated Illumina Hybridization Oven so that the top logo faces you.

You can stack up to three chambers per row for a total of six chambers. Make sure that the feet of the top chamber fit into the indents on the bottom chamber.

Chamber in Illumina Hybridization Oven

5. Incubate at 48°C for 16–24 hours.
6. Store RA1 at -25°C to -15°C for use the next day.

Resuspend XC4 to prepare for the Extend and Stain BeadChips step.

1. Add 330 ml fresh 100% EtOH to the XC4 bottle.

The resulting volume is ~350 ml.

2. On the XC4 bottle label, record that the EtOH has been added.
3. Vigorously shake to resuspend. If needed, vortex at 1625 rpm to complete suspension.
4. Leave the bottle upright on the lab bench overnight.
5. [Optional] Store at 2°C to 8°C and use up to six times over a period of two weeks.