This protocol is an extension to: Nat. Protoc. 6, 870–895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011
Cycling 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.
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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.
The authors declare no competing financial interests.
Integrated supplementary information
F121-9 FACS sorting gate.
(A.) Good library (B.) Remaining adaptor dimers around 150 bp (C.) Over-amplification (peak at 2x size). C reproduced courtesy of Agilent Technologies, Inc., from Bioanalyzer Applications for NextGenSequencing: Updates and Tips (http://www.mbl.edu/jbpc/files/2014/05/Bioanalyzer_for_NGS_slideshow.pdf), © Agilent Technologies, Inc. 2011.
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Marchal, C., Sasaki, T., Vera, D. et al. Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq. Nat Protoc 13, 819–839 (2018). https://doi.org/10.1038/nprot.2017.148
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