title: hMeDIP-3
organism: Mus musculus
description: Protocols: 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. 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). 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) For each immunoprecipitation, 1 µg of sheared gDNA was used as input. DIPs were performed according to the MagMeDIP-seq Package V2 protocol (Diagenode, C02010041), using either the provided anti-5mC antibody for MeDIP or 0.6 µg per IP of the mouse monoclonal anti-hmC antibody (Diagenode, C15200200) for hMeDIP. Additionally, a control IP with 0.6 µg of mouse IgG (Diagenode, C15400001) was included. Illumina libraries were prepared according to the Diagenode MeDIP-seq library preparation protocol. Final library concentrations were quantified using the Quantus Fluorometer (Promega) and fragment size distributions were assessed with a TapeStation (Agilent Technologies) using a D1000 ScreenTape. Libraries were sequenced on an Illumina NovaSeq X-25B instrument using paired-end 2x 150 bp reads with a target depth of 50 million reads per sample.
attributes.tag: ENA first public
attributes.value: 2026-04-14
attributes.tag: INSDC center name
attributes.value: Max-Planck-Institute for Molecular Physiology
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15961:hMeDIP-3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: ES-E14 cell
attributes.tag: cell type
attributes.value: mouse embryonic stem cell
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: mouse embryo stage
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: sample name
attributes.value: E-MTAB-15961:hMeDIP-3
attributes.tag: scientific_name
attributes.value: Mus musculus