biostudies-arrayexpressAllegra AngeloniSminthopsis crassicaudatahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16076DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. Following fertilisation, DNA methylation inherited from gametes must be reprogrammed to establish totipotency and enable the parental-to-zygotic transition. To accomplish this, non-mammalian vertebrates such as zebrafish and medaka subtly reprogram maternal 5mC profiles while maintaining high methylation levels throughout embryogenesis. In contrast, eutherian mammals such as mouse and human undergo global 5mC erasure in both embryonic and extraembryonic lineages. However, while embryonic 5mC is rapidly re-established to high levels upon implantation, the trophectoderm, which gives rise to the placenta, displays sustained and conserved DNA hypomethylation, suggesting that this drastic 5mC erasure may be functionally linked to complex placentation in mammals. To clarify whether extensive post-fertilisation 5mC erasure co-evolved with placentation, we explored embryonic methylome dynamics in another lineage of placental mammals, the marsupials. To address this, we produced detailed DNA methylation maps of embryonic development for an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), using enzymatic-methyl sequencing (Vaisvila et al Genome Res 2021).biostudies-arrayexpressSequencing - Libraries were sequenced on the Illumina NovaSeq X Plus platform (150bp, paired-end).Nucleic Acid Extraction - gDNA was extracted in biological replicate from dunnart embryonic samples using the QIAamp DNA Investigator Kit according to manufacturer instructions, and dunnart adult liver tissue using the QIAGEN DNeasy Blood & Tissue Kit according to manufacturer instructions. gDNA was isolated from sperm using the QIAGEN DNeasy Blood & Tissue Kit according to manufacturer instructions with modifications. Briefly, snapfrozen sperm in 100μL PBS was incubated with equal volume of Buffer X2 (20 mM Tris·Cl pH 8.0, 20 mM EDTA, 200 mM NaCl, 4% SDS, 80mM DTT, 12.5μl/ml QIAGEN Proteinase K), for one hour at 56°C at 300rpm. 200μL of Buffer AL and 200μL 100% ethanol were added to the sample, followed by centrifugation at 8000rpm for 1 minute in a DNeasy Mini spin column. 500μL Buffer AW1 was added to the sample which was then centrifuged at 8000rpm for 1 minute. 500μL Buffer AW2 was added to the sample which was then centrifuged at 14000rpm for 3 minutes. Sperm gDNA was then eluted in Buffer AE.Library Construction - Whole-genome methylation libraries were generated using NEBNext Enzymatic Methyl-seq (New England Biolabs) according to manufacturer instructions. For each sample, unmethylated lambda DNA and methylated pUC19 DNA were spiked-in at 0.5% and 0.025% gDNA w/w, respectively. gDNA was then sonicated to an average length of 300 base pairs in the Covaris M220 Focused Ultrasonicator using the following settings: peak incident power, 50 W; duty factor, 20%; cycles per burst, 200; treatment time, 75 s. Sonicated DNA was concentrated in a vacuum centrifuge concentrator to a final volume of 50 µL, followed by DNA end-repair and dA-tailing, adaptor ligation, TET2 oxidation of 5mC and 5hmC, APOBEC deamination of unmodified cytosine to uracil, and PCR amplification. Library size and consistency was determined by the Agilent 4200 Tapestation system and libraries were quantified using the KAPA Library Quantification Kit according to manufacturer instructions.Sample Collection - Tissues were collected from a long term breeding colony of fat-tailed dunnarts at Macquarie University. All animal work was conducted with approval from the Macquarie University Animal Ethics Committee (ARA: 2020/019). Staged embryos were collected by daily weighing of paired females through the breeding season to identify the weight changes associated with ovulation. Once at the right day of gestation, females were humanely killed with an intra-peritoneal injection of Sodium Pentobarbitone. Adult tissues were collected opportunistically, either from the same pregnant females or during tissue collections for other projects. Following dissection, embryos and tissues were snap frozen in liquid nitrogen for downstream processing. For sperm collection, the cauda epididymis was dissected from adult male testes after culling. Small incisions were made and the tissue was gently pressed to release sperm. Sperm were rinsed and collected in PBS, then snap-frozen in liquid nitrogen for downstream processing.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Sequenced reads in fastq format were trimmed using Trimmomatic (trimmomatic PE -threads 24 ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 SLIDINGWINDOW:5:20 LEADING:5 TRAILING:5 MINLEN:50). The chromosome-level assembly containing the lambda and pUC19 DNA sequences was indexed using WALT makedb command followed by mapping of trimmed reads to the indexed genome (walt -sam -m 10 -t 24 -N 10000000 -L 2000). Mapped reads in SAM format were converted to BAM and indexed using SAMtools. PCR and optical duplicates were removed using Picard Tools (MarkDuplicates REMOVE_DUPLICATES=true). Reads containing at least three non-converted CpH sites (CpA, CpC, CpT) were identified and filtered from libraries using SAMtools and Picard FilterSamReads. Genotype and methylation bias correction was performed using MethylDackel software. The number of methylated and unmethylated calls at each genomic CpG position were determined using MethylDackel (MethylDackel extract --mergeContext --minOppositeDepth 5 --maxVariantFrac 0.5 --OT 20, 130, 20, 130 --OB 20, 130, 20, 130). Only cytosines in the CpG dinucleotide context were retained for further analyses. Enzymatic conversion efficiency was estimated from the lambda and pUC19 spike-in controls.Data Transformation - Sequenced reads in fastq format were trimmed using Trimmomatic (trimmomatic PE -threads 24 ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 SLIDINGWINDOW:5:20 LEADING:5 TRAILING:5 MINLEN:50). The chromosome-level assembly containing the lambda and pUC19 DNA sequences was indexed using WALT makedb command followed by mapping of trimmed reads to the indexed genome (walt -sam -m 10 -t 24 -N 10000000 -L 2000). Mapped reads in SAM format were converted to BAM and indexed using SAMtools. PCR and optical duplicates were removed using Picard Tools (MarkDuplicates REMOVE_DUPLICATES=true). Reads containing at least three non-converted CpH sites (CpA, CpC, CpT) were identified and filtered from libraries using SAMtools and Picard FilterSamReads. Genotype and methylation bias correction was performed using MethylDackel software. The number of methylated and unmethylated calls at each genomic CpG position were determined using MethylDackel (MethylDackel extract --mergeContext --minOppositeDepth 5 --maxVariantFrac 0.5 --OT 20, 130, 20, 130 --OB 20, 130, 20, 130). Only cytosines in the CpG dinucleotide context were retained for further analyses. Enzymatic conversion efficiency was estimated from the lambda and pUC19 spike-in controls.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XBisulfite-seqSminthopsis crassicaudataAllegra AngelonifalseEnzymatic methyl-seq of embryonic development in the fat-tailed dunnart (Sminthopsis crassicaudata)DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. Following fertilisation, DNA methylation inherited from gametes must be reprogrammed to establish totipotency and enable the parental-to-zygotic transition. To accomplish this, non-mammalian vertebrates such as zebrafish and medaka subtly reprogram maternal 5mC profiles while maintaining high methylation levels throughout embryogenesis. In contrast, eutherian mammals such as mouse and human undergo global 5mC erasure in both embryonic and extraembryonic lineages. However, while embryonic 5mC is rapidly re-established to high levels upon implantation, the trophectoderm, which gives rise to the placenta, displays sustained and conserved DNA hypomethylation, suggesting that this drastic 5mC erasure may be functionally linked to complex placentation in mammals. To clarify whether extensive post-fertilisation 5mC erasure co-evolved with placentation, we explored embryonic methylome dynamics in another lineage of placental mammals, the marsupials. To address this, we produced detailed DNA methylation maps of embryonic development for an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), using enzymatic-methyl sequencing (Vaisvila et al Genome Res 2021).2025-11-14T00:00:00Z2026-05-27T12:12:57.629Z2025-11-13T15:26:18.802ZE-MTAB-16076ERP184181EFO_0002944EFO_0004170EFO_0003753EFO_0004917EFO_0005518EFO_0003816EFO_0004184