hMeDIP-seq

DNA methylation and hydroxymethylation are important epigenetic modifications for gene expression regulation. DNA methylation typically represses gene transcription. Hydroxymethylation, on the contrary, activates gene expression or prompts DNA demethylation.

5-Hydroxymethylcytosine (5hmC) is converted from 5-methylcytosine (5mC) by a group of enzymes termed ten-eleven translocation (TET) family dioxygenases. The gold-standard bisulfite conversion technologies to study DNA methylation do not distinguish between 5mC and 5hmC. However, new approaches to mapping 5hmC genomewide have advanced rapidly, although it is unclear how the different methods compare in accurately calling 5hmC.

CD Genomics can provide two 5hmC genome-wide detection approaches:

1. Whole-genome bisulfite/oxidative bisulfite sequencing (WGBS/OxBS-seq)

2. Antibody-based immunoprecipitation and sequencing of hydroxymethylated DNA (hMeDIP-seq).

The Introduction of hMeDIP-Seq

While the precise function of 5-hmC has yet to be determined, it has been postulated that it could represent a pathway to demethylated DNA, as 5-hydroxymethylcytosine is repaired as mismatched DNA and replaced with unmethylated cytosine.  Hydroxymethylation is essential for the regulation of embryonic development, stem cell pluripotency, genomic imprinting, X-chromosome inactivation, and spermatogenesis. Abnormal hydroxymethylation of DNA often causes a variety of diseases, such as cancer and male infertility. Moreover, these abnormal DNA modifications may be inherited by offspring via the gametes, and the diseases these modifications cause could also be passed to future generations.

Hydroxymethylated DNA immunoprecipitation (hMeDIP) is an immunocapture technique in which a 5-hydroxymethylcytidine antibody is used to recognize DNA fragments containing 5-hmc, and can effectively enrich hydroxymethylated DNA fragments in genome-wide assays. This selectivity is important as most common approaches to analyze DNA methylation, such as enzymatic approaches and bisulfite conversion, are unable to distinguish between 5-hmC and 5-mC.

Library construction, high-throughput sequencing and bioinformatic analysis of these hydroxymethylated DNA fragments can provide us maps of genome-wide hydroxymethylation under particular physiological or pathological conditions.

Project Workflow

Genomic DNA was sonicated to produce DNA fragments, size selection of 300 to 500 bp fragments. Illumina barcode adapters were ligated before hMeDIP. Approximately 4 mg of adaptor-ligated gDNA were pooled together in one tube. IP buffer was used to denature and dilute the mixed DNA. The denatured DNA was incubated with anti-5mC antibody. The antibody–DNA complexes were captured and purified by beads, and the hydroxymethylated fragments were amplified using adaptor-specific primers and quantified on an Agilent 2100 Bioanalyser before cluster generation and sequencing on illumina platform according to the manufacturer's protocols.

The library is constructed as shown below:

Service Specifications

	Sample Requirements Starting amount of total DNA ≥ 3 µg Sample concentration ≥ 50 ng/µl All DNA samples are validated for purity and quantity
	Sequencing Strategy Illumina, PE150 20-30M reads per sample More than 80% of bases with a ≥Q30 quality score
	Bioinformatics Analysis We provide customized bioinformatics analysis including: Sequencing Data Quality Control Alignment against reference genome Whole genome region distribution Correlation analysis PCA analysis Immunoenriched fragment analysis Peak analysis Motif analysis Functional annotation with GO/KEGG analysis Differential expression level analysis of peak associated genes

References:
[1] Ding P, Guangzhe G, Yanqing G, et al. Vitamin C increases 5-hydroxymethylcytosine level and inhibits the growth of bladder cancer[J]. Clinical Epigenetics, 2018, 10(1):94.
[2] Li G, Qian H. Hypo-hydroxymethylation of rRNA genes in the precocious Eriocheir sinensis testes revealed using hMeDIP-seq[J]. Scientific Reports, 2017, 7(1):11237.