RNA sequencing libraries from NMT-seq of opossum blastocyst single cells

Decades of work in placental (eutherian) species have constructed a paradigm of mammalian development, wherein the genome-wide erasure of parental DNA methylation is required for embryogenesis. Whether such DNA methylation reprogramming is, in fact, conserved in other mammals is unknown. To resolve this point, we generated base-resolution DNA methylation maps in gametes, embryos and adult tissues of the opossum marsupial Monodelphis domestica, revealing extensive variations from the eutherian-derived model. In stark contrast with eutherians, the marsupial genome remains hypermethylated during the cleavage stages and in the embryo proper of the blastocyst. In the extra-embryonic trophectoderm DNA methylation is reduced, suggesting an important evolutionary conserved function for DNA hypomethylation in formation of the mammalian placenta. Furthermore, unlike in eutherians, the inactive X chromosome becomes globally DNA hypomethylated during embryogenesis. Using our DNA methylation profiles, we identify a candidate mechanism for imprinted X-inactivation in marsupials, via maternal promoter DNA methylation of the Xist-like non-coding RNA RSX11. How mammalian embryos employ DNA methylation to regulate their development is therefore more mechanistically diverse than current models can accommodate. Overall design: NMT-seq was performed on single cells isolated from blastocysts of the opossum Monodelphis domestica. RNA-seq and Bisulfite-seq libraries were generated from the same single cells.