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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References
Weber, T.S., Jaehnert, I., Schichor, C., Or-Guil, M. & Carneiro, J. Quantifying the length and variance of the eukaryotic cell cycle phases by a stochastic model and dual nucleoside pulse labelling. PLoS Comput. Biol. 10, e1003616 (2014).
Hiratani, I. et al. Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol. 6, e245 (2008).
Hiratani, I. et al. Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis. Genome Res. 20, 155–169 (2010).
Hansen, R.S. et al. Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc. Natl. Acad. Sci. USA 107, 139–144 (2010).
Pope, B.D., Hiratani, I. & Gilbert, D.M. Domain-wide regulation of DNA replication timing during mammalian development. Chromosome Res. 18, 127–136 (2010).
Ryba, T. et al. Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types. Genome Res. 20, 761–770 (2010).
Ryba, T. et al. Replication timing: a fingerprint for cell identity and pluripotency. PLoS Comput. Biol. 7, e1002225 (2011).
Rivera-Mulia, J.C. et al. Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells. Genome Res. 25, 1091–1103 (2015).
Sima, J. & Gilbert, D.M. Complex correlations: replication timing and mutational landscapes during cancer and genome evolution. Curr. Opin. Genet. Dev. 25, 93–100 (2014).
Renard-Guillet, C., Kanoh, Y., Shirahige, K. & Masai, H. Temporal and spatial regulation of eukaryotic DNA replication: from regulated initiation to genome-scale timing program. Semin. Cell Dev. Biol. 30, 110–120 (2014).
Koren, A. DNA replication timing: coordinating genome stability with genome regulation on the X chromosome and beyond. Bioessays 36, 997–1004 (2014).
Sequeira-Mendes, J. & Gutierrez, C. Links between genome replication and chromatin landscapes. Plant J. 83, 38–51 (2015).
Boulos, R.E., Drillon, G., Argoul, F., Arneodo, A. & Audit, B. Structural organization of human replication timing domains. FEBS Lett. 589, 2944–2957 (2015).
Dileep, V., Rivera-Mulia, J.C., Sima, J. & Gilbert, D.M. Large-scale chromatin structure–function relationships during the cell cycle and development: insights from replication timing. Cold Spring Harb. Symp. Quant. Biol. 80, 53–63 (2015).
Rivera-Mulia, J.C. & Gilbert, D.M. Replication timing and transcriptional control: beyond cause and effect — part III. Curr. Opin. Cell Biol. 40, 168–178 (2016).
Gilbert, D.M. Temporal order of replication of Xenopus laevis 5S ribosomal RNA genes in somatic cells. Proc. Natl. Acad. Sci. USA 83, 2924–2928 (1986).
Gilbert, D.M. & Cohen, S.N. Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle. Cell 50, 59–68 (1987).
Ryba, T., Battaglia, D., Pope, B.D., Hiratani, I. & Gilbert, D.M. Genome-scale analysis of replication timing: from bench to bioinformatics. Nat. Protoc. 6, 870–895 (2011).
Wilson, K.A., Elefanty, A.G., Stanley, E.G. & Gilbert, D.M. Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification. Cell Cycle 15, 2464–2475 (2016).
Sasaki, T. et al. Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia. Exp. Hematol. 51, 71–82.e3.
Koren, A. et al. Genetic variation in human DNA replication timing. Cell 159, 1015–1026 (2014).
Mukhopadhyay, R. et al. Allele-specific genome-wide profiling in human primary erythroblasts reveal replication program organization. PLoS Genet. 10, e1004319 (2014).
Bartholdy, B., Mukhopadhyay, R., Lajugie, J., Aladjem, M.I. & Bouhassira, E.E. Allele-specific analysis of DNA replication origins in mammalian cells. Nat. Commun. 6, 7051 (2015).
Akhtar, W. et al. Chromatin position effects assayed by thousands of reporters integrated in parallel. Cell 154, 914–927 (2013).
Pope, B.D. et al. Topologically-associating domains are stable units of replication-timing regulation. Nature 515, 402–405 (2014).
Yue, F. et al. A comparative encyclopedia of DNA elements in the mouse genome. Nature 515, 355–364 (2014).
Dixon, J.R. et al. Chromatin architecture reorganization during stem cell differentiation. Nature 518, 331–336 (2015).
Ryba, T. et al. Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia. Genome Res. 22, 1833–1844 (2012).
Rivera-Mulia, J.C. et al. Allele-specific control of replication timing and genome organization during development. Preprint at. bioRxiv, https://doi.org/10.1101/221762 (2017).
Hansen, R.S., Canfield, T.K., Lamb, M.M., Gartler, S.M. & Laird, C.D. Association of fragile X syndrome with delayed replication of the FMR1 gene. Cell 73, 1403–1409 (1993).
Gilbert, D.M. Evaluating genome-scale approaches to eukaryotic DNA replication. Nat. Rev. Genet. 11, 673–684 (2010).
Koren, A. & McCarroll, S.A. Random replication of the inactive X chromosome. Genome Res. 24, 64–69 (2014).
Dileep, V. & Gilbert, D.M. Single-cell replication profiling reveals stochastic regulation of the mammalian replication-timing program. Nat. Commun., (in the press).
Dileep, V., Didier, R. & Gilbert, D.M. Genome-wide analysis of replication timing in mammalian cells: troubleshooting problems encountered when comparing different cell types. Methods 57, 165–169 (2012).
Rivera-Mulia, J.C. & Gilbert, D.M. Replicating large genomes: divide and conquer. Mol. Cell 62, 756–765 (2016).
Cornacchia, D. et al. Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells. EMBO J. 31, 3678–3690 (2012).
Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Nicol, J.W., Helt, G.A., Blanchard, S.G., Raja, A. & Loraine, A.E. The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics 25, 2730–2731 (2009).
Kent, W.J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).
Weddington, N. et al. ReplicationDomain: a visualization tool and comparative database for genome-wide replication timing data. BMC Bioinformatics 9, 530 (2008).
Rasmussen, T.P. et al. Messenger RNAs encoding mouse histone macroH2A1 isoforms are expressed at similar levels in male and female cells and result from alternative splicing. Nucleic Acids Res. 27, 3685–3689 (1999).
N2B27 Medium with LIF and 2i Inhibitors. Cold Spring Harb. Protoc. 2017 http://dx.doi.org/10.1101/pdb.rec096149 (2017).
R Development Core Team. R: A language and environment for statistical computing (R Foundation for Statistical Computing, 2005).
Bolstad, B.M., Irizarry, R.A., Åstrand, M. & Speed, T.P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193 (2003).
Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Lee, P.Y., Costumbrado, J., Hsu, C.-Y. & Kim, Y.H. Agarose gel electrophoresis for the separation of DNA fragments. J. Vis. Exp. 62, 3923 (2012).
Yokochi, T. et al. G9a selectively represses a class of late-replicating genes at the nuclear periphery. Proc. Natl. Acad. Sci. USA 106, 19363–19368 (2009).
Takebayashi, S. et al. Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains. Epigenetics Chromatin 6, 42 (2013).
Pope, B.D. et al. DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy. PLoS ONE 6, e27413 (2011).
Stamatoyannopoulos, J.A. et al. An encyclopedia of mouse DNA elements (mouse ENCODE). Genome Biol. 13, 418 (2012).
Fernandez-Vidal, A. et al. A role for DNA polymerase θ in the timing of DNA replication. Nat. Commun. 5, 4285 (2014).
Hadjadj, D. et al. Characterization of the replication timing program of 6 human model cell lines. Genomics Data 9, 113–117 (2016).
Morishima, A., Grumbach, M.M. & Taylor, J.H. Asynchronous duplication of human chromosomes and the origin of sex chromatin. Proc. Natl. Acad. Sci. USA 48, 756–763 (1962).
Takagi, N., Sugawara, O. & Sasaki, M. Regional and temporal changes in the pattern of X-chromosome replication during the early post-mplantation development of the female mouse. Chromosoma 85, 275–286 (1982).
McKenna, A. et al. Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science 353, aaf7907 (2016).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
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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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.
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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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DOI: https://doi.org/10.1038/nprot.2017.148
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