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Optimized Repli-seq: improved DNA replication timing analysis by next-generation sequencing

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Abstract

The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type–specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, the precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs, and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high-resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.

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Data availability

The datasets generated in this study are available in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), under the number GSE196749.

Abbreviations

ACF:

Autocorrelation function

BrdU:

5-Bromodeoxyuridine

FACS:

Fluorescence-activated cell sorting

IP:

Immunoprecipitation

NGS:

Next-generation sequencing

PI:

Propidium Iodide

RT:

DNA replication timing

Repli-seq:

Genome-wide analysis of RT by next-generation sequencing

HGPS:

Hutchinson-Gilford progeria syndrome

WS:

Werner syndrome

RTS:

Rothmund–Thomson syndrome

BS:

Bloom syndrome

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Acknowledgements

We thank Laura Niedernhofer and Hai Dang Nguyen for providing the MEFs and U2OS cell lines and Silvia Meyer-Nava and Anala Shetty for critically reading the manuscript.

Funding

The research reported in this study was supported by NIH R35GM137950 and U54AG076041 to JCRM.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN, USA

    Juan Carlos Rivera-Mulia, Claudia Trevilla-Garcia & Santiago Martinez-Cifuentes

  2. Stem Cell Institute, University of Minnesota Medical School, Minneapolis, MN, USA

    Juan Carlos Rivera-Mulia

  3. Masonic Cancer Center, University of Minnesota Medical School, Minneapolis, MN, USA

    Juan Carlos Rivera-Mulia

Authors
  1. Juan Carlos Rivera-Mulia
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  2. Claudia Trevilla-Garcia
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  3. Santiago Martinez-Cifuentes
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Contributions

JCRM conceived and designed the study. JCRM, CTG, and SMC conducted experiments. JCRM analyzed data and wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Juan Carlos Rivera-Mulia.

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The authors declare no competing interests.

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Responsible Editor: Lucia Carbone

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Rivera-Mulia, J.C., Trevilla-Garcia, C. & Martinez-Cifuentes, S. Optimized Repli-seq: improved DNA replication timing analysis by next-generation sequencing. Chromosome Res 30, 401–414 (2022). https://doi.org/10.1007/s10577-022-09703-7

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  • DOI: https://doi.org/10.1007/s10577-022-09703-7

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