Convert DNA

This step bisulfite converts genomic DNA samples using the Zymo EZ-96 DNA Methylation Lightning Kit. The bisulfite-converted genomic DNA (BCD) samples are then transferred to the BCD plate.

Make sure to follow the manufacturer instructions.

A minimum of 250 ng fresh DNA is supported for this reaction. Using more DNA, from 500 ng to 1000 ng, results in higher reproducibility.

• Zymo EZ-96 DNA Methylation Lightning Kit (Manual catalog # D5046 (4x96 rxns) or D5047 (8x96 rxns), automated catalog # D5049 (1x96 rxns))
• 96-well 0.2 ml skirted microplate (1–3 plates)
• Genomic DNA (for HTS Methylation)
• Use the Zymo EZ-96 DNA Methylation Lightning MagPrep kit for bisulfite conversion of genomic DNA (gDNA). For specifics, contact Illumina Technical Support.
• The conversion reagent is photosensitive. Minimize exposure to light.

The following diagram illustrates the bisulfite conversion workflow.

Each bisulfite conversion process requires the following:

• gDNA (> 250 ng)
• Elution buffer (25 µl)
1. Prepare the conversion reagent according to the manufacturer instructions for immediate use.
2. Prepare the wash buffer according to the manufacturer instructions.
3. Apply a BCD barcode label to each new 96-well 0.2 ml skirted microplate.

Use the instructions in the Zymo EZ DNA Methylation-Lightning Kit to do the following steps:

1. Add 130 µl Lightning Conversion Reagent to 20 µl DNA sample in a conversion plate.
2. Incubate in a thermal cycler using the following settings:
• 98°C for 8 minutes
• 54°C for 1 hour
3. [Optional] Hold DNA at 4°C for up to 20 hours in the thermal cycler.
1. Prepare the collection plate with the provided 600 µl M-binding buffer and 10 µl MagBinding beads.
2. Transfer and mix samples from the conversion plate to the collection plate.
3. Incubate the plate at room temperature for 5 minutes.
4. Transfer the plate to the magnetic stand. Wait 5 minutes.
5. Remove the supernatant to clean up the conversion reagent.
6. Remove the plate from the magnetic stand.
7. Add 400 µl M-Wash buffer and resuspend the beads.
8. Transfer the plate to the magnetic stand. Wait 3 minutes.
9. Remove the supernatant to clean up the M-Wash buffer.
1. Desulphonate the samples with 200 µl L-desulphonation buffer.
2. Incubate at room temperature for 15–20 minutes.
3. Transfer the plate to the magnetic stand. Wait 3 minutes.
4. Remove the supernatant to clean up the L-desulphonation buffer.
1. Add 400 µl M-Wash buffer and resuspend the beads.
2. Transfer the plate to the magnetic stand. Wait 3 minutes.
3. Remove the supernatant to clean up the M-Wash buffer.
4. Repeat steps 1–3 one time.
5. To dry the beads, transfer the plate to a heating element at 55°C for 20–30 minutes and remove residual M-wash buffer.
1. Add 25 µl elution buffer directly to the dried beads and resuspend.
2. Heat the elution at 55°C for 4 minutes.
3. Transfer the plate to a magnetic stand for 1 minute or until the beads pellet.
4. Collect the supernatant and transfer it to a clean elution plate.
1. If frozen, thaw BCD samples to room temperature and vortex to mix.
2. Apply a BCD barcode label to a new 0.8 ml MIDI plate or a new 0.2 ml TCY plate.
3. Transfer the BCD to the plate as follows.
• MIDI plate: 20 µl BCD sample to each well
• TCY plate: 10 µl BCD sample to each well

The example illustrates 96 samples. For 48 samples, fill only the blue side of the plate.

SAFE STOPPING POINT

If you are stopping, heat-seal the plate, and store at -25°C to -15°C for up to 30 days.