结合谱系追踪和单细胞染色质可及性的细胞命运决定研究

细胞的命运是如何决定的？这是生命科学领域的重要问题。单细胞组学技术的进步使人们得以深入了解在表观遗传以及转录水平细胞群体存在的异质性，从而理解一些潜在的细胞命运决定因素。谱系追踪技术的发展使人们能够对细胞谱系进行精准的追溯。谱系追踪和单细胞测序结合使得我们在发育谱系树上绘制染色质可及性和基因表达动态图谱成为可能。目前结合谱系追踪和单细胞转录组测序的细胞命运决定研究已经取得了巨大的进展，但仍不能十分清晰地阐释细胞命运决定。表观遗传层面的异质性对细胞命运决定的影响仍然不是很清楚，因此结合谱系追踪和单细胞染色质可及性测序对细胞命运决定的研究仍具有重大意义。

这里我们建立了一种结合特殊条形码标记（Cell barcoding）谱系追踪策略和单细胞染色质可及性测序（scATAC-seq）的方法，可以用于绘制细胞命运决定过程中染色质可及性动态变化图谱。通过逆转录病毒将条形码标记整合到细胞基因组，以在细胞中拷贝数较高的条形码RNA作为遗传标记保证绝大多数的单细胞都能被克隆追踪。同时，基于流式分选的scATAC-seq能够产生具有高复杂性的ATAC文库。通过这种方法在单个细胞中生成配对的条形码信息和染色质可及性信息。最后，我们将这种方法应用于分析结直肠癌细胞对于低氧环境的不同应答，研究结直肠癌细胞在低氧处理前后染色质可及性的差异。此外，此方法还适用于基于液滴的高通量scATAC-seq测序，未来可以为更复杂的组织绘制命运决定的染色质可及性动态轨迹，具有巨大的应用潜力。

How is the fate of cells determined? This is a vital question in the life sciences. Single cell omics technologies have provided a new insight to understand the underlying fate-determining signals of cells at the epigenetic and transcriptional level. Innovations combining lineage tracing and single-cell sequencing have helped revealing the heterogeneity of human cell populations, facilitated the mapping of clonal relationships between ancestors and progeny. Here, we establish a method that integrates lineage tracing and single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) to map chromatin dynamics during cell fate determination. In this method, we performed in-situ reverse transcription & ATAC-seq to generate paired genetics barcode information and chromatin landscape in single cells. To do so, genetic barcode was designed and integrated into the genome via lentivirus. In-situ reverse transcription was used to capture barcode RNAs that to improve the efficiency of cellular barcoding. “Tagmentation” of chromatin were then performed to single cells or nuclei, which were isolated with flow cytometry. Afterwards, multi-complexity scATAC library with genetic barcoding was generated. Using this method, we will analyze accessible chromatin states of colorectal cells that produce different fate decision under hypoxia. Furthermore, our approach could also be adapted to high-throughput droplet-based single-cell sequencing platform to tackle the complexity of tissues.

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