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Genome-wide chromatin analysis in mature mouse and human spermatozoa

A Corrigendum to this article was published on 11 March 2014

This article has been updated


At the end of mammalian spermatogenesis, chromatin in differentiating germ cells is extensively remodeled, with the majority of nucleosomes being removed and ultimately exchanged by highly basic proteins named protamines. Residual nucleosomes are, to various degrees, retained at regulatory sequences in human and mouse sperm. Moreover, certain histone variants and modifications remain present in regulatory sequences of subsets of genes in spermatozoa, providing opportunities for paternal inheritance of chromatin states and epigenetic control of gene expression in the subsequent generation. Here we describe in detail a method that enables the generation of soluble chromatin samples from mouse and human spermatozoa within 1 d. These samples are amendable to chromatin immunoprecipitation and high-throughput sequencing of nucleosome-associated genomic DNA, which require several additional days. We also provide computational scripts that allow straightforward analysis of large genome-wide data sets by biologists with limited computational experience. This protocol will facilitate studies of mechanisms of chromatin remodeling and epigenetic reprogramming during spermatogenesis and of paternal epigenetic inheritance. Similarly, it will help in the study of the causes of human male infertility.

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Figure 1: Schematic overview of the protocol.
Figure 2: Optimization of MNase treatment for sperm chromatin isolation (Steps 29–34).
Figure 3: Comparison of nucleosome enrichments of mouse sperm obtained by qPCR versus high-throughput sequencing analyses.
Figure 4: Analysis of H3K27me3-enriched regions in mouse sperm.
Figure 5: Reproducibility of chromatin isolation and ChIP experiments in mouse sperm.

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Gene Expression Omnibus

Change history

  • 26 February 2014

     In the version of this article initially published, the amount of N-ethylmaleimide to be dissolved was incorrect; it should be 25 mg rather than 0.25 mg. It was also not specified that the N-ethylmaleimide solution should be 1 M. In addition, the catalog number from Sigma-Aldrich for sodium acetate was incorrectly given as S2889; it should be S8625. Also, it should have been stated that the sodium acetate required is ·3H2O. The errors have been corrected in the HTML and PDF versions of the article.


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We gratefully acknowledge J. Hofsteenge for critical advice during the development of this protocol. We thank T. Roloff (FMI functional genomics group), I. Nissen (Laboratory for Quantitative Genomics, Department of Biosystems Science and Engineering (D-BSSE), Basel) and the FMI animal facility for excellent assistance. S.E. is supported as a recipient of a Boehringer Ingelheim Fond fellowship. Research in the Stadler and Peters laboratories is supported by the Novartis Research Foundation and the Swiss Initiative in Systems Biology (Cell Plasticity - Systems Biology of Cell Differentation). In addition, the Peters laboratory receives funding from the Swiss National Science Foundation (31003A_125386 and National Research Programme NRP63—Stem Cells and Regenerative Medicine), the Japanese Swiss Science and Technology Cooperation Program, the FP7 Marie Curie Initial Training Network “Nucleosome4D” and the EMBO Young Investigator Program.

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Authors and Affiliations



M.H., S.E., M.B.S. and A.H.F.M.P. conceived of and designed the experiments. M.H., with help of S.D.-B., developed the sperm chromatin isolation protocol, whereas S.E. and M.B.S developed the computational scripts and pipeline. L.R. contributed the purification protocol for human sperm. M.B.S. also provided bioinformatics training and support. S.E., M.H., M.B.S. and A.H.F.M.P. analyzed the data. S.E., M.B.S. and A.H.F.M.P. prepared the manuscript.

Corresponding authors

Correspondence to Michael B Stadler or Antoine H F M Peters.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

GEO identifiers (XLSX 12 kb)

Supplementary Data 1

Analysis of Next-generation-sequencing data with R (PDF 965 kb)

Supplementary Data 2

R code for analysis of next generation sequencing data (TXT 5 kb)

Supplementary Data 3

R_function_definitions_used_by_the_R_code_in_Supplementary_Files_1_and_2 (TXT 15 kb)

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Hisano, M., Erkek, S., Dessus-Babus, S. et al. Genome-wide chromatin analysis in mature mouse and human spermatozoa. Nat Protoc 8, 2449–2470 (2013).

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