Cycling cells duplicate their DNA content during Sphase, 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, RTis regulated during development and is altered in diseases. Here, we describe E/LRepli-seq, an extension of our Repli-chip protocol. E/LRepli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RTby 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 DNAis immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNAfrom 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 RTallelic 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.
Bibliographical noteFunding Information:
We thank R. Didier for assistance in cell sorting. This work was supported by NIH GM083337, GM085354 and DK107965 to D.M.G. C.M. is supported by ARC French fellowship SAE20160604436.
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PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't