Elucidating the mechanisms underlying chromatin maintenance upon genome replication is critical for the understanding of how gene expression programs and cell identity are preserved across cell divisions. Here, we describe two recently developed techniques, chromatin occupancy after replication (ChOR)-seq and sister chromatids after replication (SCAR)-seq, that profile chromatin occupancy on newly replicated DNA in mammalian cells in 5 d of bench work. Both techniques share a common strategy that includes pulse labeling of newly synthesized DNA and chromatin immunoprecipitation (ChIP), followed by purification and high-throughput sequencing. Whereas ChOR-seq quantitatively profiles the post-replicative abundance of histone modifications and chromatin-associated proteins, SCAR-seq distinguishes chromatin occupancy between nascent sister chromatids. Together, these two complementary techniques have unraveled key mechanisms controlling the inheritance of modified histones during replication and revealed locus-specific dynamics of histone modifications across the cell cycle. Here, we provide the experimental protocols and bioinformatic pipelines for these methods.

阐明基因组复制时染色质维持的机制对于理解基因表达程序和细胞身份如何在细胞分裂中保持至关重要。在这里，我们描述了两种最近开发的技术，即复制后染色质占据 (ChOR)-seq 和复制后姐妹染色单体 (SCAR)-seq，它们在 5 天的工作台工作中分析了哺乳动物细胞中新复制的 DNA 的染色质占据。这两种技术共享一个共同的策略，包括对新合成的 DNA 和染色质免疫沉淀 (ChIP) 进行脉冲标记，然后进行纯化和高通量测序。ChOR-seq 定量分析组蛋白修饰和染色质相关蛋白的复制后丰度，而 SCAR-seq 区分新生姐妹染色单体之间的染色质占据。总之，这两种互补技术揭示了在复制过程中控制修饰组蛋白遗传的关键机制，并揭示了整个细胞周期组蛋白修饰的位点特异性动态。在这里，我们为这些方法提供了实验协议和生物信息学管道。