biostudies-arrayexpressMarlon Steven Zambrano MilaMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-170885-Hydroxymethyluridine (5hmU) is an epigenetic thymine modification and occurs across diverse organisms. 5hmU has been linked to cancer development and therapy resistance, yet its precise biological functions remain poorly understood. Genomic profiling of 5hmU is severely hindered by cross-reactivity with 5-hydroxymethylcytosine (5hmC), producing false positives that confound biological interpretation and prevent robust genome-wide mapping in higher eukaryotes. To address these limitations, we present hmU-seq, a highly specific enzymatic–chemical labelling and capture method for genome-wide 5hmU profiling. We validated the robustness and sensitivity of hmU-seq by mapping 5hmU dynamics across two life-cycle stages of Trypanosoma brucei. We then applied hmU-seq to mouse embryonic stem cells biostudies-arrayexpressLibrary Construction - Following rRNA depletion with the NEBNext® rRNA Depletion Kit v2 (NEB), Libraries were prepared from the depleted RNA using the NEBNext UltraExpress® RNA Library Prep Kit (NEB) for Illumina according to the manufacturer’s instructions. NGS Sequencing was performed on a NovaSeq 6000 platform (GENEWIZ) in 150 bp paired-end modeSequencing - Sequencing was performed as paired-end 150 bp reads on an Illumina NovaSeq 6000 platform (GENEWIZ).Growth Protocol - WT J1 mESCs were first described in Li et al. and originally provided by the Jaenisch laboratory (Whitehead Institute, USA). mESCs were cultivated at 37°C in water-saturated, CO2-enriched (5%) atmosphere in a2iL culture medium containing 10% (vol/vol) FBS (PAN Biotech, P30-3031), 2mM L-alanyl-L-glutamine (Sigma-Aldrich, G8541), 0.1mM β-Mercaptoethanol (Sigma-Aldrich, F63689) and 1x MEM-NEAA (Sigma-Aldrich, M71145) in DMEM (Sigma-Aldrich, D6546). 103 U/mL mLif (Sigma-Aldrich, ESG1107), 3µM CHIR99021 (Axon Medchem, 1386) and 1.5µM CGP77675 (Axon Medchem, 2097) were added to the medium directly before plating the cells. All reagents were filtered using a 0.2µM filtering unit before use. Culture dishes were coated for 10-60min with 0.2% (wt/vol) gelatine (Sigma-Aldrich, G2500Sample Collection - Cells were routinely passaged when reaching 70-80% confluency by washing with DPBS (Sigma-Aldrich, D8662) and detaching the cells using TrypLE (Thermo Scientific, 12604013).Nucleic Acid Extraction - Total RNA from three independent biological replicates per condition was isolated using the Quick-RNA Miniprep Kit (Zymo Research, Cat. No. R1054)OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesData Transformation - RAW fastq file (NA normalization)Sequence Alignment - Cleaned reads were mapped to the Mus musculus reference genome (mm10) using Bowtie2.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAMus musculusMarlon Steven Zambrano MilaFranziska TraubeSusanne KramerAngela SimeoneSidney BeckerfalsehmU-Seq Reveals Genome-Wide Distribution of 5-Hydroxymethyluracil via Selective Enzymatic and Chemical Labeling5-Hydroxymethyluridine (5hmU) is an epigenetic thymine modification and occurs across diverse organisms. 5hmU has been linked to cancer development and therapy resistance, yet its precise biological functions remain poorly understood. Genomic profiling of 5hmU is severely hindered by cross-reactivity with 5-hydroxymethylcytosine (5hmC), producing false positives that confound biological interpretation and prevent robust genome-wide mapping in higher eukaryotes. To address these limitations, we present hmU-seq, a highly specific enzymatic–chemical labelling and capture method for genome-wide 5hmU profiling. We validated the robustness and sensitivity of hmU-seq by mapping 5hmU dynamics across two life-cycle stages of Trypanosoma brucei. We then applied hmU-seq to mouse embryonic stem cells 2026-09-30T00:00:00Z2026-09-30T01:00:41.312Z2026-05-26T13:13:40.968ZE-MTAB-17088ERP193969E-MTAB-16683E-MTAB-16684EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184