Integrative analysis of multi-omics layers at single cell level is critical for accurate dissection of cell-to-cell variation within certain cell populations. Here we report scCAT-seq, a technique for simultaneously assaying chromatin accessibility and the transcriptome within the same single cell. We show that the combined single cell signatures enable accurate construction of regulatory relationships between cis-regulatory elements and the target genes at single-cell resolution, providing a new dimension of features that helps direct discovery of regulatory patterns specific to distinct cell identities. Moreover, we generate the first single cell integrated map of chromatin accessibility and transcriptome in early embryos and demonstrate the robustness of scCAT-seq in the precise dissection of master transcription factors in cells of distinct states. The ability to obtain these two layers of omics data will help provide more accurate definitions of “single cell state” and enable the deconvolution of regulatory heterogeneity from complex cell populations.

Figure 1Derivation and characterization of cloned piglets from piPSCs. (A) Preimplantation and post-implantation development of the cloned embryos from piPSCs. Embryos at two-cell (a), four-cell (b), eight-cell (c), blastocyst stages (d, e) and two 36 day-old cloned fetuses (f) are shown. Scale bars are 100 μm. (B) The morphology and fluorescence of the hooves (left), tails (middle) and fibroblasts (right) of the 36 day-old embryos. Scale bars are 100 μm. (C) The morphology, fluorescence and hematoxylin/eosin-stained sections of tissues from piglet 00536-3#. Scale bars are 100 μm. (D) Cloned piglets. 00507-4# from differentiated iPF4-2 cell, 4 days old; 227-1#, 2#, 3# from undifferentiated iPF4-2, 2 days old. (E) Porcine ear fibroblasts (PEFs) from 00507-4#, EGFP positive. Scale bars are 100 μm. (F) PCR demonstrating genomic integration of Oct4, Sox2, and EGFP using tissues of the cloned fetuses and piglets. PEF, the original fibroblasts used to create iPF4-2. 00518, 00536, 00507, 227, foster mothers. 00518-1#, 00518-2#, the cloned fetuses derived from differentiated iPF4-2 cells. 00536-3# , 00507-4#, the cloned piglets derived from iPF4-2-differentiated cells. 227-1#~4#, the HMC piglets derived from iPF4-2. (G) Microsatellite analysis of the donor piPSC line iPF4-2, cloned fetuses and piglets. 00518, 00536, 00507, 227, foster mothers; 00518-1# and 00518-2#, cloned fetuses; 00536-3# and 00507-4#, the cloned piglets from differentiated iPF4-2 cells; 227-1#~4#, the cloned piglets derived from the Scriptaid-treated NT embryos from iPF4-2 cells.www.cell-research.com | Cell Research Nana Fan et al. 165npg

28Integrative analysis of multi-omics layers at single cell level is critical for accurate dissection 29 of cell-to-cell variation within certain cell populations. Here we report scCAT-seq, a 30 technique for simultaneously assaying chromatin accessibility and the transcriptome within 31 the same single cell. We show that the combined single cell signatures enable accurate 32 construction of regulatory relationships between cis-regulatory elements and the target 33 genes at single-cell resolution, providing a new dimension of features that helps direct 34 discovery of regulatory patterns specific to distinct cell identities. Moreover, we generated 35 the first single cell integrated maps of chromatin accessibility and transcriptome in human 36 2 pre-implantation embryos and demonstrated the robustness of scCAT-seq in the precise 1 dissection of master transcription factors in cells of distinct states during embryo 2

BackgroundInvestigating cell fate decision and subpopulation specification in the context of the neural lineage is fundamental to understanding neurogenesis and neurodegenerative diseases. The differentiation process of neural-tube-like rosettes in vitro is representative of neural tube structures, which are composed of radially organized, columnar epithelial cells and give rise to functional neural cells. However, the underlying regulatory network of cell fate commitment during early neural differentiation remains elusive.ResultsIn this study, we investigated the genome-wide transcriptome profile of single cells from six consecutive reprogramming and neural differentiation time points and identified cellular subpopulations present at each differentiation stage. Based on the inferred reconstructed trajectory and the characteristics of subpopulations contributing the most toward commitment to the central nervous system lineage at each stage during differentiation, we identified putative novel transcription factors in regulating neural differentiation. In addition, we dissected the dynamics of chromatin accessibility at the neural differentiation stages and revealed active cis-regulatory elements for transcription factors known to have a key role in neural differentiation as well as for those that we suggest are also involved. Further, communication network analysis demonstrated that cellular interactions most frequently occurred in the embryoid body stage and that each cell subpopulation possessed a distinctive spectrum of ligands and receptors associated with neural differentiation that could reflect the identity of each subpopulation.ConclusionsOur study provides a comprehensive and integrative study of the transcriptomics and epigenetics of human early neural differentiation, which paves the way for a deeper understanding of the regulatory mechanisms driving the differentiation of the neural lineage.

Spermatogenic lineage has been directly generated in spermatogonial stem cell (SSC) conditions from human pluripotent stem cells (PSCs). However, it remains unknown whether mouse embryonic stem cells (ESCs) can directly differentiate into advanced male germ cell lineage in the same conditions. Here, we showed rather low efficiency of germ-like cell generation from mouse ESCs in SSC conditions. Interestingly, addition of retinoic acid (RA) into SSC conditions enabled efficient differentiation of mouse ESCs into germ-like cells, as shown by the activation of spermatogenesis-associated genes such as Mvh, Dazl, Prdm14, Stella, Scp1, Scp3, Stra8 and Rec8. In contrast, for cells cultured in control medium, the activation of the above genes barely occurred. In addition, RA with SSC conditions yielded colonies of Acrosin-expressing cells and the positive ratio reached a peak at day 6. Our work thus establishes a simple and cost-efficient approach for male germ like cell differentiation from mouse PSCs and may propose a useful strategy for studying spermatogenesis in vitro.