Introduction

Methylation of cytosines can mediate epigenetic gene silencing and is the most prominent DNA modification in eukaryotes. MeDIP is an immunocapturing approach to enrich DNA that is methylated. The principle is that genomic DNA is randomly sheared by sonication and immunoprecipitated with an antibody that specifically recognizes 5-methylcytidine (5mC) (Figure 1). This protocol has been used to generate comprehensive DNA methylation profiles on a genome scale in mammals and plants[1-3], and to identify abnormally methylated genes in cancer cells [1].

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Procedure

Preparation of genomic DNA

The MeDIP protocol has been successfully used with genomic DNA from various organisms that contain methylated cytosines in their genome (human, mouse, Arabidopsis thaliana , Neurospora crassa ).

1. Resuspend the cell pellet or the homogenized tissue in 300 µl TE in a 2 ml eppendorf tube (the volume should be increased for big cell pellets or tissue samples)
2. Add 300 µl Lysis buffer containing 20 µl proteinase K (10 mg/ml stock) (note 1)
3. Incubate at 55°C for at least 5 hours
4. Extract with 1 volume phenol (600 µl)
5. Extract with 1 volume chloroform (600 µl)
6. Precipitate the DNA with 2 volumes ethanol 75 mM NaAcetate (1.2 ml)
7. Resuspend the DNA pellet in TE containing 20 µg/ml RNAse A (note 2)

Sonication of genomic DNA

Genomic DNA is randomly sheared by sonication to generate fragments between 300 and 1000 bp. Genomic DNA can also be fragmented with restriction enzymes like Alu I, but it is not recommended for unbiased microarray studies. The sonication efficiency varies with DNA concentration, sonicator settings and size and quality of the sonication tip, therefore it is recommended to systematically check the size of the sheared DNA to ensure equal sonication between experiments.

1. Dilute the genomic DNA in TE in a 1.5 ml eppendorf tube (10-20 µg DNA in 400 µl TE, 40-60 µg DNA in 700 µl TE)
2. Sonicate 5 times 10 seconds (BRANSON digital Sonifier model 450, used with the tapered Microtip, amplitude 20%), with 1 minute intervals between pulses (keep the tube on ice during the sonication)
3. Load 5 µl on an agarose gel to check the size of the DNA (mean size should be 300-1000 bp) (Figure 2)
4. If necessary, sonicate one or two additional pulses until the size of the DNA is 300-1000 bp
5. Precipitate the sonicated DNA with 400 mM NaCl, glycogen (1 µl) and 2 volumes 100% ethanol
6. Resuspend the DNA pellet in water and measure DNA concentration

Immunoprecipitation of methylated DNA (MeDIP)

The sonicated DNA is then immunoprecipitated with an monoclonal antibody against 5-methylcytidine (5mC) [4] (Eurogentec #BI-MECY-1000). A portion of the sonicated DNA should be left untreated to serve as input control.

1. Dilute 4 µg (note 3) of sonicated DNA in 450 µl TE
2. Denature for 10 minutes in boiling water and immediately cool on ice for 10 minutes
3. Add 51 µl of 10x IP buffer
4. Add 10 µl of 5mC Antibody (note 4)
5. Incubate 2 hours at 4°C with overhead shaking
6. Pre-wash 40 µl of Dynabeads with 800 µl PBS-BSA 0.1% for 5 minutes at RT with shaking (comment 1)
7. Collect the beads with a magnetic rack and repeat wash with 800 µl PBS-BSA 0.1%
8. Collect the beads with a magnetic rack and resuspend in 40 µl of 1x IP buffer
9. Add Dynabeads to the sample (note 5)
10. Incubate 2 hours at 4°C with overhead shaking
11. Collect the beads with a magnetic rack and wash with 700 µl 1x IP buffer for 10 minutes at RT with shaking
12. Repeat wash with 700 µl 1x IP buffer twice
13. Collect the beads with a magnetic rack and resuspend in 250 µl Proteinase K digestion buffer
14. Add 7 µl proteinase K (10 mg/ml stock)
15. Incubate 3 hours at 50°C (use a shaking heating block 800rpm to prevent sedimentation of the beads) (comment 2)
16. Extract with 1 volume phenol (250 µl)
17. Extract with 1 volume chloroform (250 µl)
18. Precipitate the DNA with 400 mM NaCl (20 µl NaCl 5M), glycogen (1 µl) and 2 volumes 100% ethanol (500 µl)
19. Resuspend the DNA pellet in 60 µl TE and keep at �20°C (note 6)