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MeDIP Kit Overview
The MeDIP Kit is designed to immunoprecipitate and enrich single-stranded DNA (ssDNA) fragments containing 5-methylcytosine (5-mC). The MeDIP Kit contains a monoclonal 5-methylcytosine antibody, a bridging antibody, and the necessary buffers to perform methylated DNA immunoprecipitation. The high specificity and affinity of the antibody enables separation of 5-mC DNA from DNA containing 5-hmC and other DNA methylation variants using single-stranded input DNA.
Active Motif's fast, magnetic protocol has been streamlined to minimize the number of wash and incubation steps, saving you valuable time. The kit also includes MseI-digested human genomic DNA and PCR primers that can be used to verify the efficiency of the enrichment. Additionally, the kit contains a bar magnet for easy separation and elution of the enriched methylated DNA.
The high affinity of the 5-mC antibody used in the MeDIP Kit enables detection of methylated cytosines regardless of their context. This means that methylation does not need to occur in the context of a CpG dinucleotide, unlike methyl-binding protein (MBD) affinity enrichment methods which requires CpG methylation for proper detection.
MeDIP Highlights
- Specific antibody only enriches 5-mC methylated DNA
- Includes bridging antibody to increase efficiency of DNA capture
- Optimized magnetic protocol to minimize hands-on time
- Includes positive control human genomic DNA and PCR primers to verify efficiency
FlowChart of MeDIP
Figure 1: Flow chart of the MeDIP Method.
Single-stranded, fragmented genomic DNA containing 5-methylcytosine can be specifically captured from the rest of the genomic DNA with the monoclonal 5-mC antibody in the presence of a bridging antibody and magnetic beads. Following an overnight incubation, the enriched DNA is then eluted from the beads.
MeDIP Kit Contents
*Protein G magnetic beads are shipped on dry ice and will arrive frozen. DO NOT refreeze the magnetic beads after their first use. Once thawed, the Protein G magnetic beads should be stored at 4°C.
- 5-methylcytosine mAb (1 μg/μl), 20 μg, Store at -20°C
- Bridging Antibody (1 μg/μl), 500 μl, Store at -20°C
- Mouse IgG (1 μg/μl), 100 μl, Store at 4°C
- Protease Inhibitor Cocktail (PIC), 100 μl, Store at -20°C
- Buffer C, 10 ml, Store at -20°C
- Buffer D, 10 ml, Store at -20°C
- Elution Buffer AM2, 1.6 ml, Store at -20°C
- Neutralization Buffer, 1.6 ml, Store at -20°C
- Human genomic DNA, MseI digested (20 ng/μl), 250 μl, Store at -20°C
- ZC3H13 PCR Primer Mix (2.5 μM). 400 μl, Store at -20°C
- Protein G magnetic beads*, 250 μl, Store at 4°C
- Sterile water, 1 ml, Store at RT
- 0.2 ml PCR tubes, 1 pack, Store at RT
- Bar magnet and glue dots, 1 ea, Store at RT
MeDIP Kit Data
The MeDIP Kit is able to selectively enrich for DNA fragments containing 5-methylcytosine DNA methylation from the rest of the genomic DNA population. The MeDIP Kit was tested using 500 ng of the included MseI-digested human genomic DNA in the presence or absence of bridging antibody. Eluted DNA was purified with Active Motif's Chromatin IP DNA Purification Kit (Catalog No. 58002) and tested in real time PCR with the included ZC3H13 PCR primer set. Results shown in figure 2 demonstrate the importance of using a bridging antibody within the assay. In figure 3, enriched DNA was tested using either the included positive control ZC3H13 PCR primer mix or a negative control GAPDH PCR primer mix.
Figure 2: MeDIP Kit in the presence or absence of bridging antibody.
MseI-digested human genomic DNA (500 ng) was processed using the MeDIP Kit with the 5-methylcytidine mAb or negative control mouse IgG in the presence or absence of bridging antibody. Eluted DNA was purified and tested using real time PCR with the included ZC3H13 PCR primer mix. The 5-methylcytidine antibody specifically enriched methylated DNA in the presence of bridging antibody, but did not enrich for DNA with the mouse IgG or in the absence of bridging antibody. The ZC3H13 locus analyzed in this experiment is methylated in human genomic DNA and therefore should amplify. Data shown are the results from samples assayed in duplicate.
Figure 3: Real time PCR results of the control DNA with ZC3H13 and GAPDH PCR primer sets.
MseI-digested human genomic DNA (500 ng) was processed using the MeDIP Kit with the 5-methylcytosine mAb or negative control mouse IgG. Eluted DNA was column purified with Active Motif's Chromatin IP DNA Purification Kit (Catalog No. 58002) and tested using real time PCR with the included ZC3H13 PCR primer mix and a negative control GAPDH PCR primer mix. The 5-methylcytosine antibody specifically enriched methylated DNA with the ZC3H13 locus but did not enrich for DNA with either the GAPDH locus or the mouse IgG. Data shown are the results from samples assayed in duplicate.
Figure 4: Active Motif’s MeDIP Kit can be used to enrich for methylation in cfDNA.
Serum samples were collected in serum separator tubes (SST) from two healthy “normal” adults, one <25 yo and one >60 yo. Cell-Free DNA (cfDNA) was purified with the Active Motif Cell-Free DNA Purification Kit (Cat no. 25503/25504) and quantified with a Qubit fluorometer. Undigested cfDNA (200 ng) was processed using the Active Motif MeDIP Kit (Cat no. 55009) with the 5-mC Ab or negative control mouse IgG Ab in the kit. Eluted DNA was purified and tested using real time PCR with the included ZC3H13 PCR primer set. The 5-methylcytidine antibody specifically enriched methylated DNA but did not enrich for DNA with the mouse IgG. The ZC3H13 locus analyzed in this experiment is methylated in human genomic DNA and therefore should amplify.
Figure 5: Example DNA Libraries from the Active Motif MeDIP Kit.
The Swift Biosciences Accel-NGS™ 1S PLUS DNA Library Kit, now the IDT xGen ssDNA & Low-Input DNA Library Preparation Kit (Cat# 10009859), was used to prepare libraries after using the Active Motife MeDIP Kit with MseI digested gDNA and undigested cfDNA.
Figure 6: MeDIP-Seq uncovers aging-associated differential methylation patterns in cfDNA.
Serum samples were collected in serum separator tubes (SST) from healthy “normal” adults, two <25 yo and two >60. Cell-Free DNA (cfDNA) was purified with the Active Motif Cell-Free DNA Purification Kit (Cat no. 25503/25504) and quantified with a Qubit fluorometer. 200 ng cfDNA was used in the MeDIP Kit and libraries were prepared using the Swift Biosciences Accel-NGS™ 1S PLUS DNA Library Kit, now the IDT xGen ssDNA & Low-Input DNA Library Preparation Kit (Cat# 10009859). Input to library prep was determined using AluI quantification. MeDIP and Input libraries were sequenced (38bp PE chemistry on Next-Seq500) and processed as per Active Motif’s standard pipeline for MeDIP-Seq. Peaks called using MACS2 relative to input (p-value cutoff = 1.00x10-7).
Figure 7: NGS data generated using Active Motif’s MeDIP Kit correlates well with CpG density.
DNA was enriched from 1 ug of adaptor-ligated human PBMC DNA using Active Motif’s MeDIP Kit. NGS was performed using the Illumina platform to generate 26 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows that the enriched regions (purple peaks) correlate well with CpG density (tan peaks).
Figure 8: NGS data generated using Active Motif’s MeDIP Kit detects methylation at CpG shores.
DNA was enriched from 1 ug of adaptor-ligated human PBMC DNA using Active Motif’s MeDIP Kit. NGS was performed using the Illumina platform to generate 26 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows two examples of DNA methylation being detected at CpG shores rather than in the CpG island itself. This data agrees with recent findings showing that a large portion of methylated sites occur in these regions that are adjacent to CpG islands.
MeDIP Kit FAQs
How should I prepare libraries for MeDIP-seq?
Because the antibody used for MeDIP requires single-stranded fragments, the DNA must be denatured prior to immunoprecipitation. As a result, library prep protocols which are designed for double-stranded DNA, will not work. There are two options recommended for library preparation for MeDIP-Seq when using this kit. One option is to use a library prep kit designed for single-stranded DNA after the immunoprecipitation protocol. Our recommendation is the the xGen™ ssDNA & Low-Input DNA Library Prep Kit (IDT Catalog no. 10009859). Alternatively, a double-stranded library prep kit can be used by ligating the adapters to the fragmented double-stranded DNA prior to performing the immunoprecipitation (MeDIP) protocol and followed by library amplification after MeDIP.
What sequencing parameters should I used for MeDIP?
We recommend single-end, 75 bp read length with 30 million reads.
Should I use MeDIP or MethylCollector MBD Capture on my DNA samples?
It depends. The MethylCollector MBD Capture Kit uses a His-tagged recombinant MBD2b protein which specifically binds CpG methylated DNA fragments for enrichment. The MeDIP kit uses a methylation specific antibody for enrichment. The advantage of MeDIP over MBD capture is that it covers both CpG and non CpG sites. The disadvantage is that MeDIP requires more input DNA (100 ng) than MBD capture (5 ng), requires single-stranded DNA, and because it’s antibody based, can have a higher background than enzymatic based methods.
How much control gDNA should I expect post-IP?
We do not recommend quantifying the yield (post-IP DNA) by nanodrop or Qubit as it will be a very low concentration and not accurate. Instead, perform a qPCR reaction using the provided ZC3H13 primer pair to determine the percent input recovered for a methylated locus in the DNA. To calculate percent input, compare the anti-5-mC precipitated DNA versus input sample DNA. See the manual for more guidance.
What type of coverage can I expect from MeDIP?
Typically ~88% of CpG sites.
Can I purchase the 5-mC antibody separately?
Yes, the antibody is cat# 39649.
MeDIP Kit Publications
Search our database of customer publications that have used our MeDIP Kit.
MeDIP Kit Documents
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| Name | Format | Cat No. | Price | |
|---|---|---|---|---|
| MeDIP | 10 rxns | 55009 | $480 | Add to Cart |
