biostudies-arrayexpressAllegra AngeloniSminthopsis crassicaudatahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16075DNA 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 single-cell DNA methylation maps of the bilaminar blastocyst for an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata).biostudies-arrayexpressLibrary Construction - Single cell bisulfite sequencing (scBS-seq) libraries were prepared as described with minor modifications (Clark et al Nature Protocols 2017). First, RNA was removed from single cell lysates using oligo-dT conjugated magnetic beads. Genomic DNA was then purified using a 0.8X volume of AMPure XP beads (Beckman Coulter) and subjected to bisulfite conversion using the EZ Methylation Direct Kit (Zymo). The oligo used in preamplification was modified to be compatible with NEBNext index primers used later in the protocol, and to prevent self-amplification: /5SpC3/CTACACGACGCTCTTCCGATCTNNNNNN (IDT, HPLC purified). After completing 5 rounds of preamplification and exonuclease treatment, the first-strand synthesis products were cleaned-up using a 0.65X volume of AMPure XP beads. Adaptor 2 tagging was then performed, and a 0.65X volume of buffer from AMPure XP beads was used to purify the second-strand synthesis products. Library amplification was then performed using NEBNext Multiplex Oligos for Illumina (Dual Index Primers) and 1x NEBNext Q5 Ultra II Master Mix (New England Biolabs), as follows: 98°C for 30 s; 14 repeats of 98°C for 10 s, 65°C for 30 s, 65°C for 45 s; 65°C for 5 min; 4°C hold. Amplified scBS-seq libraries were cleaned-up using a 0.65X volume of buffer from AMPure XP beads, assessed using the TapeStation High Sensitivity D5000 ScreenTape assay (Agilent), and quantified using Qubit dsDNA High Sensitivity assay (Thermo Fisher Scientific)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. Following dissection, embryos were snap frozen in liquid nitrogen for downstream processing.Sequencing - Indexed libraries were pooled in equimolar amounts for sequencing on the Illumina NovaSeq X Plus platform (150bp, paired-end).Nucleic Acid Extraction - Single nuclei from 3 snap frozen E9 embryos were isolated using The S2 Genomics Singulator Platform, with the Low Volume Nuclei Isolation V2 protocol according to manufacturer instructions. DAPI-positive nuclei were sorted in 96-well plates containing 2.5μL of Buffer RLT (QIAGEN) and 1U/µL RNAse inhibitor using BD FACSAria™ III Cell Sorter. Plates were immediately placed on dry ice, sealed, and stored at -80°C until library preparation.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequenced reads in fastq format were trimmed using TrimGalore (trim_galore --cores 4 --paired --clip_r1 6 --clip_r2 6) and read alignment to the chromosome-level assembly was performed using Bismark (bismark --non_directional --unmapped --multicore 2), with reads not meeting paired-end constraints mapped in single-end format. Mapped reads in BAM format were deduplicated using Bismark (deduplicate_bismark --bam --multiple), and per-CpG methylation levels were called using Bismark bismark_methylation_extractor (--gzip --bedGraph --paired-end/--single-end) and bismark2bedGraph (default settings) functions.Sequence Alignment - Sequenced reads in fastq format were trimmed using TrimGalore (trim_galore --cores 4 --paired --clip_r1 6 --clip_r2 6) and read alignment to the chromosome-level assembly was performed using Bismark (bismark --non_directional --unmapped --multicore 2), with reads not meeting paired-end constraints mapped in single-end format. Mapped reads in BAM format were deduplicated using Bismark (deduplicate_bismark --bam --multiple), and per-CpG methylation levels were called using Bismark bismark_methylation_extractor (--gzip --bedGraph --paired-end/--single-end) and bismark2bedGraph (default settings) functions.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XBisulfite-seqSminthopsis crassicaudataAllegra AngelonifalseSingle-cell bisulfite sequencing of fat-tailed dunnart (Sminthopsis crassicaudata) E9 pooled embryosDNA 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 single-cell DNA methylation maps of the bilaminar blastocyst for an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata).2025-11-14T00:00:00Z2026-05-27T01:01:43.746Z2025-11-13T15:30:10.251ZE-MTAB-16075ERP184199EFO_0002944EFO_0004170EFO_0003753EFO_0004917EFO_0005518EFO_0003816EFO_0004184