title: Enzymatic methyl-seq of embryonic development in the fat-tailed dunnart (Sminthopsis crassicaudata)
design: Enzymatic methyl-seq of embryonic development in the fat-tailed dunnart (Sminthopsis crassicaudata)
library_construction_protocol: 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. 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.    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.