We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing, allowing discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method reveals novel associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes [1][2][3][4][5] . Global epigenetic reprogramming accompanies these changes [6][7][8] , but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe the first single cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by TET-mediated demethylation, and a concomitant increase of accessibility. In striking contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled prior to cell fate decisions, providing the molecular logic for a hierarchical emergence of the primary germ layers.Recent technological advances have enabled the profiling of multiple molecular layers at single cell resolution 9-13 , providing novel opportunities to study the relationship between the transcriptome and epigenome during cell fate decisions. We applied scNMT-seq (singlecell Nucleosome, Methylome and Transcriptome sequencing 12 ) to profile 1,105 single cells isolated from mouse embryos at four developmental stages (Embryonic Day (E) 4.5, E5.5, E6.5 and E7.5) which comprise the exit from pluripotency and primary germ layer specification (Figure 1a-d, Extended Data Fig. 1). Cells were assigned to a specific lineage by mapping their RNA expression profiles to a comprehensive single-cell atlas 4 from the same stages, when available, or using marker genes (Extended Data Fig. 2). By performing Argelaguet et al.

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

Primary, symptomatic HIV-1 infection is associated with high titers of cytopathic, replication-competent viral strains, and during such infection potential infectivity is enhanced. Effective control of HIV-1 replication during primary infection implies the activation of clinically important mechanisms of immune defense that merit further examination in relation to the development of antiviral therapy and vaccines.

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a novel single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.