Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq

Abstract

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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Figure 1: Overview of Repli-seq protocol and analysis.
Figure 2: Quantile normalization and Loess smoothing allow comparison between samples.
Figure 3: Repli-chip and Repli-seq give highly similar replication-timing profiles at a genome-wide level.
Figure 4: Repli-seq allows the discrimination between haplotypes.

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Acknowledgements

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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Contributions

D.M.G., C.M. and T.S. conceived the study and designed the experiments. T.S., K.W., J.S., C.T.-G., C.N., E.N. and J.C.R.-M. performed wet experiments. D.V., J.S. and C.M. devised the computational methods. C.M., T.S. and D.M.G. wrote the manuscript.

Corresponding author

Correspondence to David M Gilbert.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 FACS gating strategy.

F121-9 FACS sorting gate.

Supplementary Figure 2 Representative Bioanalyzer results from library quality control.

(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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Supplementary Figures 1 and 2, Supplementary Table 1, Supplementary Data 1 and 2, and the Supplementary Methods. (PDF 661 kb)

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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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