Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

Abstract

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

An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure

“…Whole-Genome Single-Cell 5mC Sequencing (scMspJI-Seq) Samples were prepared as described in scMspJI-seq with minor modifications (Sen et al, 2019). TRA-1-85-positive single hPGCLCs we sorted into each reaction well of a 384-well plate containing 4 mL of Vapor-Lock (QIAGEN) and 200 nL of lysis buffer (0.0875% IGEPAL CA-630) and were stored at À80 C until use.…”

“…TRA-1-85-positive single hPGCLCs we sorted into each reaction well of a 384-well plate containing 4 mL of Vapor-Lock (QIAGEN) and 200 nL of lysis buffer (0.0875% IGEPAL CA-630) and were stored at À80 C until use. Cells were prepared as described by Sen et al (2019) with some minor adjustment in reagent volumes. The ds-adaptor sequences used are described in scMspJI-seq (Sen et al, 2019).…”

“…Cells were prepared as described by Sen et al (2019) with some minor adjustment in reagent volumes. The ds-adaptor sequences used are described in scMspJI-seq (Sen et al, 2019). Excluding the Vapor-Lock, all reagent dispenses were performed using the Nanodrop II liquid handling robot (BioNex Solutions).…”

“…The samples were vacuum centrifuged to a volume of 6.4 mL, and 9.6 mL of in vitro transcription mix was added (1.6 mL of each ribonucleotide, 1.6 mL of T7 buffer, 1.6 mL T7 enzyme mix [MEGAscript T7 Transcription Kit, Ambion]) and incubated at 37 C for 13 h. Library preparation steps were performed as described previously (Mooijman et al, 2016;Rooijers et al, 2019). The identification of 5 mC sites in the human genome from the sequencing data is described in scMspJIseq (Sen et al, 2019).…”

“…MspJI recognizes 5 mC sites and cuts gDNA 16 bp downstream of the site leaving a 4-bp 5 0 overhang, allowing both position-specific and strand-specific detection of 5 mC. The strand-specific distribution of 5 mC on a chromosome is quantitatively described by a metric called strand bias, as described previously (Sen et al, 2019;Mooijman et al, 2016). Ninety-six U2 D4 hPGCLCs and 94 U2 D4C10 hPGCLCs cells were sequenced, along with 2 negative control empty reaction wells.…”