Protein-DNA interactions are critical to the regulation of gene expression, but it remains challenging to define how cell-to-cell heterogeneity in protein-DNA binding influences gene expression variability. Here we report a method for the simultaneous quantification of protein-DNA contacts by combining single-cell DNA adenine methyltransferase identification (DamID) with mRNA sequencing of the same cell (scDam&T-seq). We apply scDam&T-seq to reveal how genome-lamina contacts or chromatin accessibility correlate with gene expression in individual cells. Furthermore, we provide single-cell genome-wide interaction data on a Polycomb-group protein, RING1B, and the associated transcriptome. Our results show that scDam&T-seq is sensitive enough to distinguish mouse embryonic stem cells cultured under different conditions and their different chromatin landscapes. Our method will enable analysis of protein-mediated mechanisms that regulate cell type-specific transcriptional programs in heterogeneous tissues.

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The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis.

Protein-DNA interactions are essential to establish cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DamID and CEL-Seq2. DamID works through the tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity of the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, on the other hand, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription (IVT). scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 days, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 days. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a protein of interest. It can be performed in any laboratory with access to FACS, robotic and high-throughput sequencing facilities.

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