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.
Subscribe to Journal
Get full journal access for 1 year
only $41.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
A custom script is included as Supplementary Data 4.
Egger, G., Liang, G. N., Aparicio, A. & Jones, P. A. Epigenetics in human disease and prospects for epigenetic therapy. Nature 429, 457–463 (2004).
Campos, E. I. & Reinberg, D. Histones: annotating chromatin. Annu. Rev. Genet. 43, 559–599 (2009).
Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41–45 (2000).
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).
Zhao, Y. & Garcia, B. A. Comprehensive catalog of currently documented histone modifications. Cold Spring Harb. Perspect. Biol. 7, a025064 (2015).
Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).
Wang, Z. et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat. Genet. 40, 897–903 (2008).
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).
Adli, M., Zhu, J. & Bernstein, B. E. Genome-wide chromatin maps derived from limited numbers of hematopoietic progenitors. Nat. Methods 7, 615–618 (2010).
Shankaranarayanan, P. et al. Single-tube linear DNA amplification (LinDA) for robust ChIP-seq. Nat. Methods 8, 565–567 (2011).
Lara-Astiaso, D. et al. Immunogenetics. Chromatin state dynamics during blood formation. Science 345, 943–949 (2014).
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).
Rotem, A. et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat. Biotechnol. 33, 1165–1172 (2015).
Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, e21856 (2017).
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).
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).
Kaya-Okur, H. S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930 (2019).
Harada, A. et al. A chromatin integration labelling method enables epigenomic profiling with lower input. Nat. Cell Biol. 21, 287–296 (2019).
Consortium, E. P. et al. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).
Roadmap Epigenomics Consortium. et al. Integrative analysis of 111 reference human epigenomes. Nature 518, (317–330 (2015).
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).
Thorsen, T., Maerkl, S. J. & Quake, S. R. Microfluidic large-scale integration. Science 298, 580–584 (2002).
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).
Brower, K., White, A. K. & Fordyce, P. M. Multi-step variable height photolithography for valved multilayer microfluidic devices. J. Vis. Exp. 2017, 55276 (2017).
Ma, S. et al. Cell-type-specific brain methylomes profiled via ultralow-input microfluidics. Nat. Biomed. Eng. 2, 183–194 (2018).
Horz, W. & Altenburger, W. Sequence specific cleavage of DNA by micrococcal nuclease. Nucleic Acids Res. 9, 2643–2658 (1981).
Dingwall, C., Lomonossoff, G. P. & Laskey, R. A. High sequence specificity of micrococcal nuclease. Nucleic Acids Res. 9, 2659–2673 (1981).
Jung, Y. L. et al. Impact of sequencing depth in ChIP-seq experiments. Nucleic Acids Res. 42, e74 (2014).
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).
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).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137–R137 (2008).
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).
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).
Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
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).
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.
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.
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.
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
A LabVIEW program for operating the single-unit MOWChIP device.
A LabVIEW program for operating the eight-unit MOWChIP device.
A LayoutEditor file for the eight-unit MOWChIP device.
A custom script for ChIP-seq data analysis.
A video on major steps involved in MOWChIP.
About this article
Cite this article
Zhu, B., Hsieh, Y., 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
Analytical Chemistry (2020)