Repli-seq Sample Preparation using Cell Sorting with Cell-Permeant Dyes.
Repli-seq Sample Preparation using Cell Sorting with Cell-Permeant Dyes.
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DOI：
10.1002/cpz1.945
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发表时间：
2023-11
期刊：
影响因子：
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通讯作者：
Rivera-Mulia, Juan Carlos
中科院分区：
文献类型：
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作者：
Meyer-Nava, Silvia;Shetty, Anala V;Rivera-Mulia, Juan Carlos
复制时间与基因表达和染色质组织显着相关，在细胞分化过程中动态变化，并在疾病状态下发生改变。通过 Repli-seq 在活跃复制的细胞中进行复制时序的全基因组分析。目前的 Repli-seq 方法需要固定大量细胞。对于对固定敏感或数量非常有限的样品类型来说，这是一个障碍。在本文中，我们概述了用于 Repli-seq 处理活细胞和完整细胞核的优化方法。我们的协议可以处理每个样品的较少数量的细胞，并减少处理时间和样品损失，同时获得高质量的数据。此外，对于容易形成团块且难以解离成单细胞悬浮液的样品，我们还概述了 Repli-seq 细胞核的分离、染色和处理方法。从活细胞和完整细胞核获得的 Repli-seq 数据与从标准方案获得的数据相当。基本方案：活细胞分离和染色 替代方案：细胞核分离和染色
Replication timing is significantly correlated with gene expression and chromatin organization, changes dynamically during cell differentiation, and is altered in diseased states. Genome-wide analysis of replication timing is performed in actively replicating cells by Repli-seq. Current methods for Repli-seq require cells to be fixed in large numbers. This is a barrier for sample types that are sensitive to fixation or are in very limited numbers. In this article, we outline optimized methods to process live cells and intact nuclei for Repli-seq. Our protocol enables the processing of a smaller number of cells per sample and reduces processing time and sample loss while obtaining high-quality data. Further, for samples that tend to form clumps and are difficult to dissociate into a single-cell suspension, we also outline methods for isolation, staining, and processing of nuclei for Repli-seq. The Repli-seq data obtained from live cells and intact nuclei are comparable to those obtained from the standard protocols. Basic Protocol: Live cell isolation and staining Alternate Protocol: Nuclei isolation and staining
影响因子：
3.7
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通讯作者：
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