Comprehensive Mapping of Transcriptionally-active Enhancers During Murine Cardiogenesis at Single Cell Resolution

An in-depth understanding of the complex mechanisms that orchestrate normal embryonic heart development is required to disambiguate the origin of congenital heart disease. However, the epigenetic landscapes governing cell type-specific gene expression patterns during heart development at single cell resolution has yet to be revealed. Here we use simultaneous single cell RNA and assay for transposase accessible chromatin sequencing to construct a comprehensive multimodal map of embryonic murine heart development, profiling cell type- and timepoint-specific epigenetic regulatory elements as well as their gene targets. Furthermore, we compare these regulatory elements with published GWAS hits to map variants implicated in key developmental processes and clinical phenotypes to enhancers for genes that are actively transcribed during cardiac development. Taken together, our work provides a rich resource for the broader scientific community that empowers future studies in heart development and disease and offers a simple computational framework for integrating and profiling multiomic data in single cells. Mouse embryonic hearts were harvested at embryonic days 8.5, 10.5, and 16.5 and dissociated into single cells. Nuclei were extracted from cell suspensions and underwent ATAC transposition followed by single nuclei capture using the 10X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev F) protocol. ScRNA and scATAC libraries were generated using the 10X Genomics protocol. Subsequent sequencing was conducted using an Illumina NOvaSeq 6000 at 20,000 read pars per nuclei for the gene expression library and 25,000 read pairs per nuclei for the ATAC library.要厘清先天性心脏病的发病起源，需深入解析调控正常胚胎心脏发育的复杂分子机制。然而，当前在单细胞分辨率下，心脏发育过程中调控细胞类型特异性基因表达模式的表观遗传图谱仍未被完全揭示。本研究采用单细胞RNA测序与转座酶可及性染色质测序（assay for transposase accessible chromatin sequencing, ATAC-seq）联合分析技术，构建了小鼠胚胎心脏发育的综合多模态图谱，系统解析了细胞类型与时间点特异性的表观遗传调控元件及其靶基因。本研究团队进一步将上述调控元件与已发表的全基因组关联研究（Genome-Wide Association Study, GWAS）显著位点进行比对，将参与关键发育过程与临床表型的遗传变异映射至心脏发育阶段活跃转录基因的增强子区域。综上，本研究为全球科研共同体提供了丰富的高质量研究资源，可为未来心脏发育与疾病相关研究提供重要支撑，同时也为单细胞多组学数据的整合分析提供了简便的计算分析框架。研究人员分别在小鼠胚胎发育第8.5、10.5及16.5天采集胚胎心脏组织，将其解离为单细胞悬液。从细胞悬液中提取细胞核，进行ATAC转座反应，随后采用10X Genomics Chromium Next GEM单细胞多组学ATAC+基因表达（CG000338 Rev F）实验流程完成单细胞核捕获。基于10X Genomics配套实验流程构建单细胞RNA（scRNA）与单细胞ATAC（scATAC）测序文库。后续测序工作采用Illumina NovaSeq 6000平台完成，其中基因表达文库的测序深度为每个细胞核20000条测序读段对，ATAC文库的测序深度为每个细胞核25000条测序读段对。