25 26 DNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from 27 the parental genomes during preimplantation mammalian development is critical to reset 28 the methylome of terminally differentiated gametes to the pluripotent cells in the 29 blastocyst. While active and passive modes of demethylation have both been suggested 30 to play a role in this process, the relative contribution of these two mechanisms to 31 genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput 32 single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, 33 thereby allowing us to systematically probe the dynamics of global demethylation. First, 34 when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell 35 strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric 36 2 levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of 37 maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, 38 we discovered that methylation maintenance is active till the 16-cell stage followed by 39 passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage 40 of development. Finally, we found that human preimplantation embryos qualitatively 41 show temporally delayed yet similar demethylation dynamics as mouse preimplantation 42 embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and 43 cost-effective method to map the strand-specific genome-wide patterns of 5mC in single 44 cells, thereby enabling quantitative investigation of methylation dynamics in 45 developmental systems. 46 47In mammalian systems, DNA methylation (5-methylcytosine or 5mC) is a key epigenetic

DNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells.