{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Marlon Zambrano Mila"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15857"],"description":["5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are the two main epigenetic modifications of mammalian DNA, and play crucial roles in cell differentiation, development, and tumorigenesis. Both modifications co-exist with unmodified cytosine in palindromic CpG dyads in different strand-symmetric and -asymmetric combinations, each having unique regulatory potential. To facilitate investigating the individual functions of such dyad modifications, we here report HM-DyadCap. This method employs MECP2 HM – an evolved methyl-CpG-binding domain of the mC reader MECP2 – for the simple capture and sequencing of DNA fragments containing the CpG dyad hmC/mC that frequently occurs in mouse embryonic stem cell and brain genomes. In vitro binding studies reveal a high discrimination of MECP2 HM against diverse off-target dinucleotides in vitro. We conduct comparative mapping experiments for mESC genomes with HM-DyadCap, standard MethylCap based on wild type MECP2, as well as the antibody-based MeDIP and hMeDIP protocols. We find that MECP2 HM is blocked by hmC glucosylation, and conduct control enrichments with glucosylated genomes that indicate highly selective enrichment of hmC/mC dyads by MECP2 HM. Metagene profiles correlate hmC/mC with actively transcribed genes, and reveal global enrichment in gene bodies as well as depletion at transcription start sites. We anticipate that HM-DyadCap will enable effective mapping of hmC/mC with implications for biomarker discovery and unravelling the function of this dyad in diverse aspects of chromatin biology."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Growth Protocol - E14tg2a mouse embryonic stem cells were grown in dishes pre-coated with 0.1 % gelatin (w/vol, Sigma) in GMEM supplemented with 10% fetal bovine serum, sodium pyruvate, 50 μM β-mercaptoethanol, glutamax, non-essential amino acids (all from Gibco/ThermoFisher) and 10 ng/ml murine leukemia inhibitory factor (LIF, Protein Expression Facility, MPI Dortmund).","Library Construction - Dyad Cap libraries were prepared using the NEBNext Ultra II DNA Library Preparation Kit. DNA purification and size selection was carried out using NEBNext Sample Purification Beads according to the protocol with bead volumes corresponding to a target fragment size of 200 bp. Fragment size distribution was verified using a TapeStation.","Sequencing - Illumina NovaSeq X - Illumina","Sample Collection - Cells were passaged every 2-3 days to maintain cultures between 10% and 90% confluency. For analysis, near-confluent cultures were released from culture vessels with trypsin, spun down, and snap-frozen in liquid nitrogen before further processing.","Nucleic Acid Extraction - gDNA was isolated from E12tg2a mESCs using the Monarch Genomic DNA Purification Kit (NEB, T3010) according to the manufacturer’s instructions. gDNA was sheared to an average fragment size of 200 bp using a Bioruptor Pico sonication device (Diagenode)"],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - Initial quality assessment of raw reads was carried out using FastQC, followed by adapter trimming and removal of low-quality bases using Trim Galore. The cleaned reads were then aligned to the Mus musculus reference genome mm10 using Bowtie2.  Retaining only properly paired reads and removing PCR duplicates was done using Samtools. To identify enriched regions, peak calling was performed on deduplicated BAM files using MACS2.","Data Transformation - Counts Per Million (CPM) normalization"],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq X"],"study_type":["MBD-seq"],"species":["Mus musculus"],"pubmed_authors":["Daniel Summerer","Marlon Zambrano Mila","Lena Engelhard","Sidney Becker"],"additional_accession":[]},"is_claimable":false,"name":"HM-DyadCap – Capture and Mapping of Asymmetric 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads in Mammalian DNA","description":"5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are the two main epigenetic modifications of mammalian DNA, and play crucial roles in cell differentiation, development, and tumorigenesis. Both modifications co-exist with unmodified cytosine in palindromic CpG dyads in different strand-symmetric and -asymmetric combinations, each having unique regulatory potential. To facilitate investigating the individual functions of such dyad modifications, we here report HM-DyadCap. This method employs MECP2 HM – an evolved methyl-CpG-binding domain of the mC reader MECP2 – for the simple capture and sequencing of DNA fragments containing the CpG dyad hmC/mC that frequently occurs in mouse embryonic stem cell and brain genomes. In vitro binding studies reveal a high discrimination of MECP2 HM against diverse off-target dinucleotides in vitro. We conduct comparative mapping experiments for mESC genomes with HM-DyadCap, standard MethylCap based on wild type MECP2, as well as the antibody-based MeDIP and hMeDIP protocols. We find that MECP2 HM is blocked by hmC glucosylation, and conduct control enrichments with glucosylated genomes that indicate highly selective enrichment of hmC/mC dyads by MECP2 HM. Metagene profiles correlate hmC/mC with actively transcribed genes, and reveal global enrichment in gene bodies as well as depletion at transcription start sites. We anticipate that HM-DyadCap will enable effective mapping of hmC/mC with implications for biomarker discovery and unravelling the function of this dyad in diverse aspects of chromatin biology.","dates":{"release":"2026-04-13T00:00:00Z","modification":"2026-04-13T08:18:30.221Z","creation":"2025-10-24T15:14:16.272Z"},"accession":"E-MTAB-15857","cross_references":{"ENA":["ERP182928"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0003750","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184"]}}