Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
Center for Genomics and Personalized Medicine, Florida State University, Tallahassee, Florida, USA.
Nat Protoc. 2018 May;13(5):819-839. doi: 10.1038/nprot.2017.148. Epub 2018 Mar 29.
This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.
Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011 在 S 期，循环细胞会根据一个名为复制时间（RT）的特定程序复制其 DNA 含量。早期和晚期复制区域在突变率、转录活性、染色质标记和亚核位置方面存在差异。此外，RT 在发育过程中受到调控，并在疾病中发生改变。在这里，我们描述了 E/L Repli-seq，这是我们的 Repli-chip 方案的扩展。E/L Repli-seq 是一种通过下一代测序（NGS）快速、稳健且相对廉价的分析 RT 的方法，允许全面评估细胞过程与 RT 的联系。简而言之，用 BrdU 脉冲标记细胞，然后通过流式细胞术对早期和晚期 S 期分数进行分选。从这两个分数中免疫沉淀标记的新生 DNA，并对其进行测序。数据处理生成一个包含早期和晚期 S 期分数中新生 DNA 比值的单个 bedGraph 文件。结果与 Repli-chip 相当，但具有全基因组序列信息和增加的动态范围的额外优势。我们还提供了用于下游分析的计算流程，用于使用单核苷酸多态性（SNP）解析分相基因组以分析 RT 等位基因异步，以及直接与 Repli-chip 数据进行比较。该方案可在测序前最多 3 天内完成，需要基本的细胞和分子生物学技能，以及对 Unix 和 R 的基本了解。
