MeDIP Application Protocol

Methylated DNA Immunoprecipitation (MeDIP) is an antibody-based technique used to capture and enrich the methylated fraction of a DNA sample. Subsequent to MeDIP, DNA methylation can be analyzed with a variety of downstream applications, including PCR, ChIP, and next-generation sequencing.

MeDIP Protocol.

A typical MeDIP assay can be completed within 2 days and involves the following fundamental steps, using isolated and purified genomic DNA as input material:

1. Fragmentation of the DNA sample.
2. Complexation of the methylated DNA fraction with an antibody that is specific for the particular methylation of interest (e.g., 5-mC, 5-hmC) and is not cross-reactive with other methylated forms.
3. Release of methylated DNA from the antibody complex.
4. Purification of the methylated DNA.

1) Genomic DNA Shearing

1. Dilute DNA to 0.5-8 µg per 80 µl of deionized water in a 1.5 ml microfuge tube.
2. Using a Fisher 700 W sonic dismembrator (attached to a cooling chamber) set to 20% amplitude, sonicate with pulses of 1 s on/off for a total of 2 series 110 s in the presence of ice in the cooling chamber, replenishing it in between series.

The conditions of DNA shearing should be optimized based on the sonicator equipment. If desired, remove 4 µl (300 ng) of sonicated DNA for agarose gel analysis. The length of the DNA fragments should be 200-800 bps.

2) Methylated DNA/Antibody Complexation

1. Add 330 µl of TE buffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA) to 70 µl (5.25 µg) of sonicated DNA.
2. Incubate at 95°C for 10 min, then immediately place on ice for 5 min.
3. Add 100 µl of 5X IP buffer to the denatured sonicated DNA. Note: 100 ml of 5X IP buffer may be prepared by mixing 50 mL of 100 mM sodium phosphate buffer (pH 7.0), 14 ml of 5 M NaCl, 250 µl of Triton X-100, and 35.75 ml of DNase-free water. Mix well and filter-sterilize with a 0.2 µm pore size filter.
4. Add 5 µg (2.5 µl) of the appropriate antibody.
5. Incubate at 4°C overnight on a rotating platform.

Rotate at a low enough speed to avoid significant foaming.

3) Methylated DNA Release

1. Transfer 80 µl of resuspended Protein A/G Plus agarose beads to a 1.5 ml microfuge tube. Centrifuge at 4°C for 2 min at 6000 rpm. Discard the supernatant.
2. Add 500 µl of DNA/antibody complex to the beads.
3. Incubate at 4°C for 2 h on a rotating platform.
4. Centrifuge the agarose bead-bound DNA/antibody complex at 4°C for 2 min at 6000 rpm. Discard the supernatant. Add 1 mL of 1X IP buffer and incubate at 4°C for 5 min on a rotating platform. Centrifuge at 4°C for 2 min at 6000 rpm. Discard the supernatant. Repeat this wash step two more times.
5. Resuspend the beads in 210 µL of digestion buffer. Note:100 ml of digestion buffer may be prepared by mixing 5 ml of 1 M Tris-HCl (pH 8.0), 2 ml of 0.5 M EDTA, 5 ml of 10% SDS, and 88 ml DNase-free water.
6. Add 20 µL of Proteinase K (20 mg/ml) to the resuspended beads.
7. Incubate at 55°C for 2 h on a rotating platform.

4) Methylated DNA Clean-Up

1. Filter with a Pierce spin-filtering column at RT for 30 sec at max speed. Discard the column and keep the flow through.
2. Add 3 µl of glycogen (5 mg/ml) to the flow through. Mix well.
3. Add 20 µl of ice-cold 5 M NaCl and then 750 µl of ice-cold ethanol. Mix well.
4. Incubate for 30 min, on ice or at RT, to precipitate the DNA.
5. Centrifuge at 4°C for 30 min at 14,000 rpm. Discard the supernatant.
6. Completely dry the pellet at 50°C for 5 min in a heating block.
7. Resuspend in 30 µl water. Incubate at 50°C for 5 min in the heating block and measure the DNA concentration (expected yield = 300-500 ng, 10-15 ng/µl).

The methylated DNA is now ready for use or storage at -20°C.

ChIP-grade antibodies are recommended. For 5-methylcytosine capture, EpiGentek’s highly cited 5-mC monoclonal antibody (catalog no. A-1014) has been validated for use in MeDIP assays. A normal mouse IgG should also be included as a negative control.

For a more rapid and highly efficient MeDIP protocol, EpiGentek offers the Methylamp and EpiQuik series of methylated and hydroxymethylated DNA capture kits, complete all-in-one sets of optimized reagents for the capture, enrichment, and purification of methylated DNA fragments directly from genomic DNA, cells, or tissues in only under 4 hours.

Reference
Guerrero-Bosagna C, Jensen P. Optimized method for methylated DNA immune-precipitation. MethodsX. 2015;2:432-439.

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