Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

MOWChIP-seq for low-input and multiplexed profiling of genome-wide histone modifications

Nature Protocols volume 14, pages 3366–3394 (2019)Cite this article

Subjects

Abstract

Epigenetic mechanisms such as histone modifications play critical roles in adaptive tuning of chromatin structures. Profiling of various histone modifications at the genome scale using tissues from animal and human samples is an important step for functional studies of epigenomes and epigenomics-based precision medicine. Because the profile of a histone mark is highly specific to a cell type, cell isolation from tissues is often necessary to generate a homogeneous cell population, and such operations tend to yield a low number of cells. In addition, high-throughput processing is often desirable because of the multiplexity of histone marks of interest and the large quantity of samples in a hospital setting. In this protocol, we provide detailed instructions for device fabrication, setup, and operation of microfluidic oscillatory washing–based chromatin immunoprecipitation followed by sequencing (MOWChIP-seq) for profiling of histone modifications using as few as 100 cells per assay with a throughput as high as eight assays in one run. MOWChIP-seq operation involves flowing of chromatin fragments through a packed bed of antibody-coated beads, followed by vigorous microfluidic oscillatory washing. Our process is semi-automated to reduce labor and improve reproducibility. Using one eight-unit device, it takes 2 d to produce eight sequencing libraries from chromatin samples. The technology is scalable. We used the protocol to study a number of histone modifications in various types of mouse and human tissues. The protocol can be conducted by a user who is familiar with molecular biology procedures and has basic engineering skills.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2: Schematics for electrical and pressure control systems associated with single-unit and eight-unit MOWChIP devices.
Fig. 3: The assembly of the pneumatic control system for the eight-unit MOWChIP device shown in a step-by-step manner.
Fig. 4: Design and operation of an eight-unit MOWChIP device.
Fig. 5: The experimental setup for MOWChIP operation.
Fig. 6: DNA fragment size distributions measured by a TapeStation.
Fig. 7: Optimization of antibody quantity using MOWChIP-qPCR for four histone marks.
Fig. 8: MOWChIP-seq data for H3K27me3, H3K9me3, H3K36me3 and H3K79me2 generated using 1,000 nuclei from mouse prefrontal cortex per assay.

Similar content being viewed by others

Data availability

Gene Expression Omnibus: MOWChIP-seq data on H3K27me3, H3K9me3, H3K36me3, and H3K79me2 in mouse prefrontal cortex (Fig. 8) are deposited under accession no. GSE123606.

Code availability

A custom script is included as Supplementary Data 4.

References

  1. Egger, G., Liang, G. N., Aparicio, A. & Jones, P. A. Epigenetics in human disease and prospects for epigenetic therapy. Nature 429, 457–463 (2004).

    Article  CAS  Google Scholar 

  2. Campos, E. I. & Reinberg, D. Histones: annotating chromatin. Annu. Rev. Genet. 43, 559–599 (2009).

    Article  CAS  Google Scholar 

  3. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41–45 (2000).

    Article  CAS  Google Scholar 

  4. Zhou, V. W., Goren, A. & Bernstein, B. E. Charting histone modifications and the functional organization of mammalian genomes. Nat. Rev. Genet. 12, 7–18 (2011).

    Article  Google Scholar 

  5. Zhao, Y. & Garcia, B. A. Comprehensive catalog of currently documented histone modifications. Cold Spring Harb. Perspect. Biol. 7, a025064 (2015).

    Article  Google Scholar 

  6. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).

    Article  CAS  Google Scholar 

  7. Wang, Z. et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat. Genet. 40, 897–903 (2008).

    Article  CAS  Google Scholar 

  8. Cao, Z., Chen, C., He, B., Tan, K. & Lu, C. A microfluidic device for epigenomic profiling using 100 cells. Nat. Methods 12, 959–962 (2015).

    Article  CAS  Google Scholar 

  9. Adli, M., Zhu, J. & Bernstein, B. E. Genome-wide chromatin maps derived from limited numbers of hematopoietic progenitors. Nat. Methods 7, 615–618 (2010).

    Article  CAS  Google Scholar 

  10. Shankaranarayanan, P. et al. Single-tube linear DNA amplification (LinDA) for robust ChIP-seq. Nat. Methods 8, 565–567 (2011).

    Article  CAS  Google Scholar 

  11. Lara-Astiaso, D. et al. Immunogenetics. Chromatin state dynamics during blood formation. Science 345, 943–949 (2014).

    Article  CAS  Google Scholar 

  12. Ma, S., Hsieh, Y.-P., Ma, J. & Lu, C. Low-input and multiplexed microfluidic assay reveals epigenomic variation across cerebellum and prefrontal cortex. Sci. Adv. 4, eaar8187 (2018).

    Article  Google Scholar 

  13. Rotem, A. et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat. Biotechnol. 33, 1165–1172 (2015).

    Article  CAS  Google Scholar 

  14. Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, e21856 (2017).

    Article  Google Scholar 

  15. Skene, P. J., Henikoff, J. G. & Henikoff, S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat. Protoc. 13, 1006–1019 (2018).

    Article  CAS  Google Scholar 

  16. Hainer, S. J., Boskovic, A., McCannell, K. N., Rando, O. J. & Fazzio, T. G. Profiling of pluripotency factors in single cells and early embryos. Cell 177, 1319–1329 (2019).

    Article  CAS  Google Scholar 

  17. Kaya-Okur, H. S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930 (2019).

    Article  Google Scholar 

  18. Harada, A. et al. A chromatin integration labelling method enables epigenomic profiling with lower input. Nat. Cell Biol. 21, 287–296 (2019).

    Article  CAS  Google Scholar 

  19. Consortium, E. P. et al. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

    Article  Google Scholar 

  20. Roadmap Epigenomics Consortium. et al. Integrative analysis of 111 reference human epigenomes. Nature 518, (317–330 (2015).

    Google Scholar 

  21. Brower, K. et al. An open-source, programmable pneumatic setup for operation and automated control of single- and multi-layer microfluidic devices. HardwareX 3, 117–134 (2018).

    Article  Google Scholar 

  22. Thorsen, T., Maerkl, S. J. & Quake, S. R. Microfluidic large-scale integration. Science 298, 580–584 (2002).

    Article  CAS  Google Scholar 

  23. Unger, M. A., Chou, H.-P., Thorsen, T., Scherer, A. & Quake, S. R. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288, 113–116 (2000).

    Article  CAS  Google Scholar 

  24. Brower, K., White, A. K. & Fordyce, P. M. Multi-step variable height photolithography for valved multilayer microfluidic devices. J. Vis. Exp. 2017, 55276 (2017).

  25. Ma, S. et al. Cell-type-specific brain methylomes profiled via ultralow-input microfluidics. Nat. Biomed. Eng. 2, 183–194 (2018).

    Article  CAS  Google Scholar 

  26. Horz, W. & Altenburger, W. Sequence specific cleavage of DNA by micrococcal nuclease. Nucleic Acids Res. 9, 2643–2658 (1981).

    Article  CAS  Google Scholar 

  27. Dingwall, C., Lomonossoff, G. P. & Laskey, R. A. High sequence specificity of micrococcal nuclease. Nucleic Acids Res. 9, 2659–2673 (1981).

    Article  CAS  Google Scholar 

  28. Jung, Y. L. et al. Impact of sequencing depth in ChIP-seq experiments. Nucleic Acids Res. 42, e74 (2014).

    Article  CAS  Google Scholar 

  29. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  30. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  31. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  Google Scholar 

  32. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137–R137 (2008).

    Article  Google Scholar 

  33. Xu, S., Grullon, S., Ge, K. & Peng, W. Spatial clustering for identification of ChIP-enriched regions (SICER) to map regions of histone methylation patterns in embryonic stem cells. Methods Mol. Biol. 1150, 97–111 (2014).

    Article  CAS  Google Scholar 

  34. Thorvaldsdóttir, H., Robinson, J. T. & Mesirov, J. P. Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013).

    Article  Google Scholar 

  35. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  Google Scholar 

  36. Cox, M. C., Deng, C., Naler, L., Lu, C. & Verbridge, S. S. Effects of culture condition on epigenomic profiles of brain tumor cells. ACS Biomater. Sci. Eng. 5, 1544–1552 (2019).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. González-Maeso of Virginia Commonwealth University for providing mouse brain samples. This work was supported by US National Institutes of Health grants CA214176 (C.L.), EB017235 (C.L.), HG009256 (C.L.) and HG008623 (C.L.) and seed grants from the Center for Engineered Health of the Virginia Tech Institute for Critical Technology and Applied Science.

Author information

Author notes

  1. These authors contributed equally: Bohan Zhu, Yuan-Pang Hsieh.

Authors and Affiliations

  1. Department of Chemical Engineering, Virginia Tech, Blacksburg, VA, USA

    Bohan Zhu, Yuan-Pang Hsieh, Travis W. Murphy, Qiang Zhang, Lynette B. Naler & Chang Lu

Authors
  1. Bohan Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuan-Pang Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Travis W. Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lynette B. Naler
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chang Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.L. conceived the concept of MOWChIP-seq and supervised the research and development. Y.-P.H. and B.Z. conducted MOWChIP-seq assays with various samples. T.W.M. set up the control system for multi-unit MOWChIP devices. Q.Z. helped optimize the protocol. L.B.N. wrote the script for data analysis. B.Z., T.W.M., L.B.N. and C.L. wrote the manuscript. All authors commented on the manuscript.

Corresponding author

Correspondence to Chang Lu.

Ethics declarations

Competing interests

C.L. declares that he is a co-inventor of a US patent (9,732,377) issued on MOWChIP-seq. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Jeong Heon Lee and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Cao, Z., Chen, C., He, B., Tan, K. & Lu, C. Nat. Methods 12, 959–962 (2015) https://doi.org/10.1038/nmeth.3488

Cox, M., Deng, C., Naler, L. B., Lu, C. & Verbridge, S. S. ACS Biomater. Sci. Eng. 5, 1544–1552 (2019) https://doi.org/10.1021/acsbiomaterials.9b00161

Integrated supplementary information

Supplementary Fig. 1 Various tubing assemblies used in the protocol.

(a) The tubing assemblies. (b) The assemblies connected to a microfluidic device.

Supplementary information

Supplementary Information

Supplementary Fig. 1

Reporting Summary

Supplementary Data 1

A LabVIEW program for operating the single-unit MOWChIP device.

Supplementary Data 2

A LabVIEW program for operating the eight-unit MOWChIP device.

Supplementary Data 3

A LayoutEditor file for the eight-unit MOWChIP device.

Supplementary Data 4

A custom script for ChIP-seq data analysis.

Supplementary Video 1

A video on major steps involved in MOWChIP.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhu, B., Hsieh, YP., Murphy, T.W. et al. MOWChIP-seq for low-input and multiplexed profiling of genome-wide histone modifications. Nat Protoc 14, 3366–3394 (2019). https://doi.org/10.1038/s41596-019-0223-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0223-x

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research