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5-hydroxymethylcytosine (5-hmC), like 5-methylcytosine (5-mC), is a DNA modification that occurs on cytosines and influences gene expression. Several recent studies have shown that the 5-hmC modification is enriched in the gene bodies of expressed genes although 5-hmC is present at much lower levels than 5-mC, and 5-hmC levels can vary depending on the cell type, with stem cells and brain having the highest levels of 5-hmC.
The low levels of 5-hmC in the genome can present technical challenges in experiments designed to map the modification across the entire genome. That’s why Active Motif Epigenetic Services provides a service to map 5-hmC across the genome using 5-hmC DNA immunoprecipitation (hMeDIP). This assay consists of immunoprecipitation of genomic DNA using our monoclonal 5-hmC antibody. The enriched DNA is sequenced using Illumina Next-gen sequencing technology, resulting in genome-wide maps of 5-hmC methylation.
The hMeDIP-Seq Service includes:
Customers submit purified DNA, frozen tissues or cell pellets, then we:
- Prepare the DNA sample.
- Perform hMeDIP with a 5-hmC mAb.
- Amplify the enriched DNA sample.
- Perform Next-Gen sequencing.
- Analyze the data and deliver it to the customer.
To learn more, please give us a call or send us an Epigenetic Services Information Request. You can also download Active Motif’s Epigenetic Services Profile.
Figure 1: hMeDIP-Seq performed on human brain.
hMeDIP was performed with 5-Hydroxymethylcytosine (5-hmC) antibody (Catalog No. 39999) and 2 µg of human brain DNA. Enriched DNA was sequenced on the Illumina GA II and 19 million sequence tags were mapped to identify regions enriched for 5-hmC. The image above shows an 8 Mb region on chromosome 5 with 5-hmC enrichment across several gene bodies.
Figure 2: Gene Ontology analysis of the 2,500 genes with the highest hMeDIP signals.
The 2,500 genes with the highest hMeDIP signals were selected for Gene Ontology analysis. The analysis shows that these selected genes were biased toward brain expression. In addition, the graph above shows that the other most statistically significant ontologies were also subsets of the brain. These results show that 5-hmc enrichment correlates well with expected gene expression.
