MeDIP Sequencing Protocol

DNA Shearing

1. Extract genomic DNA according to the cells or tissues studied.
2. Sonicate purified genomic DNA.
3. Dilute 6 μg genomic DNA into 130 μl (final volume) 1× TE Buffer and pipet into the appropriate Covaris tube.
4. Set Covaris to 300 bp fragment size program.
5. Run program for each tube.
6. Run 5 or 10 μl (around 400–500 ng) of sheared genomic DNA and 10 μl DNA ladder on 1.5% agarose gel to verify fragments size. Unsonicated DNA (100–200 ng) can be run on the same gel as a comparison.

Antibody Addition

1. Measure volume of sonicated DNA after gel run and dilute it with 1× TE Buffer to 400 μl.
2. Heat-denature in dry bath heating block for 10 min at 95 °C and immediately cool on ice for 10 min.
3. While keeping the sample cold, add 100 μl of cold 5× IP and 4–5 μg of antibody (monoclonal mouse anti 5-methyl-cytidine) to the denatured sonicated DNA. Incubate the DNA–antibody mixture overnight on a rotator at 4 °C.

Bind Beads to DNA–Antibody Mixture

1. Prewash magnetic beads (We use Dynabeads M-280 sheep anti-mouse IgG) as follows: Thoroughly resuspend the beads by pipetting up and down or rotating. They need to be in a homogeneous solution and if processing multiple samples they need to be resuspended frequently since they settle fast.
2. Transfer needed total volume (50 μl per sample) to a centrifuge tube, add the same volume of Washing Buffer (at least 1 ml) and resuspend.
3. Place the tube in a magnetic rack for 1–2 min and discard supernatant.
4. Resuspend beads in 1 ml Washing Buffer and incubate for 1 min on ice. Put tube on magnet for 1–2 min and discard supernatant.
5. Remove the tube from the magnetic rack and resuspend washed beads in the same volume of 1× IP Buffer as the initial volume of beads.
6. Add 50 μl of beads to the 500 μl of DNA–antibody mixture from step 2.
7. Incubate for 2 h on a rotating platform at 4 °C.

DNA–Antibody–Bead Mixture Washing

1. After the 2 h incubation wash beads three times with 1× IP Buffer as follows: Place tube in magnetic rack for 1–2 min and discard supernatant. Remove tube from magnetic rack. Add 1 ml of cold 1× IP Buffer. Mix by inverting tube or gently vortexing. Incubate tube for 1 min on ice. Place tube in magnetic rack for 1–2 min and discard the supernatant. Repeat twice for a total of three washes.
2. Resuspend the beads in 250 μl Digestion Buffer.
3. Add 3.5 μl Proteinase K (20 mg/ml) to the resuspended beads.
4. Incubate for 2–3 h on a rotating platform at 55 °C.

DNA Purification

1. Remove parafilm and add 250 μl phenol–chloroform–isoamyl alcohol to each tube. Vortex for 30 s and centrifuge at 14,000 × g for 5 min at room temperature. Remove the aqueous supernatant and transfer it to a fresh microcentrifuge tube.
2. Add 250 μl of chloroform to the supernatant from step 1. Vortex briefly and centrifuge at 14,000 × g for 5 min at room temperature. Remove the aqueous supernatant and transfer it to a fresh microcentrifuge tube.
3. Add 2 μl of the coprecipitant GlycoBlue (20 mg/ml, Life Technologies) and mix well.
4. Add 20 μl 5 M NaCl and then 500 μl of 100% ethanol. Mix well.
5. Precipitate in −20 °C freezer for 1 h to overnight.
6. Centrifuge at 14,000 × g for 20 min at 4 °C. Carefully remove the supernatant while not disturbing the blue pellet.
7. Wash once to twice with 1 ml 70% ethanol by incubating at −20 °C for 10 min then spinning again for 10 min. Discard supernatant. Then spin again briefly to collect residual liquid to bottom of tube and remove all the liquid with gel loading or other fine pipette tip.
8. Air-dry the samples on bench.
9. Resuspend in 25 μl of nuclease-free water.
10. Measure the DNA concentration.

Libraries for Next Generation Sequencing

1. Since the DNA fragments retrieved through MeDIP are single stranded, the first step needs to produce double stranded DNA for library preparation.
2. Use between 10 and 1000 ngof single-stranded DNA fragments and anneal 10 ng/μl random hexamer primers to the sample by heating it in a thermal cycler to 95 °C, then cooling immediately on ice.
3. Perform second strand DNA synthesis, which if using the NEB kit as mentioned above would be step 1.4.
4. Follow the rest of the manufacturer's protocol to receive libraries for next generation sequencing.
5. Determine the library yield with the help of Qubit High Sensitivity dsDNA Kit, then perform quality control for fragment size range and concentration/molarity.
6. Libraries are sequenced on the sequencers available to you, for example Illumina HiSeq

Reference:

1. Ben Maamar M, Sadler-Riggleman I, Beck D, et al. Genome-wide mapping of DNA methylation 5mC by methylated DNA immunoprecipitation (MeDIP)-sequencing[J]. DNA Modifications: Methods and Protocols, 2021: 301-310.