Abstract
Although conventional methods such as MNase-seq, DNase-seq, and ChIP-seq have been used effectively to assess chromatin and locus accessibility at the genome level, these techniques generally require large numbers of input cells. As such, much of what we understand in terms of epigenetic regulation and locus accessibility in CD4+ T cell subsets comes from in vitro culture systems, which allow for the production of large numbers of polarized T cells. However, obtaining such numbers directly ex vivo from tissues of individual mice is difficult. Here we describe a method combining cytokine reporter mice and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to identify genome wide locus accessibility in a small number of cytokine-expressing CD4+ T cells. This method takes you from cell isolation to library generation and quality control to query. Because the Il4 and Ifng loci are reciprocally regulated in polarized CD4+ T cell subsets (Th1 vs. Th2), we investigated the ability of this approach to identify transposase integration in both IL-4- and IFN-γ-expressing CD4+ T cells isolated directly from the lung and lymph nodes after helminth infection.
The original version of this chapter was revised. A correction to this chapter can be found at https://doi.org/10.1007/978-1-4939-7896-0_31
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Change history
05 September 2018
Correction to: R. Lee Reinhardt (ed.), Type 2 Immunity: Methods and Protocols, Methods in Molecular Biology, vol. 1799, https://doi.org/10.1007/978-1-4939-7896-0
References
Buenrostro JD, Wu B, Chang HY, Greenleaf WJ (2015) ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol 109:21.29.21–21.29.29. https://doi.org/10.1002/0471142727.mb2129s109
Lara-Astiaso D, Weiner A, Lorenzo-Vivas E, Zaretsky I, Jaitin DA, David E, Keren-Shaul H, Mildner A, Winter D, Jung S, Friedman N, Amit I (2014) Immunogenetics. Chromatin state dynamics during blood formation. Science 345(6199):943–949. https://doi.org/10.1126/science.1256271
Shih HY, Sciume G, Mikami Y, Guo L, Sun HW, Brooks SR, Urban JF Jr, Davis FP, Kanno Y, O'Shea JJ (2016) Developmental acquisition of regulomes underlies innate lymphoid cell functionality. Cell 165(5):1120–1133. https://doi.org/10.1016/j.cell.2016.04.029
Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ (2013) Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10(12):1213–1218. https://doi.org/10.1038/nmeth.2688
Seumois G, Chavez L, Gerasimova A, Lienhard M, Omran N, Kalinke L, Vedanayagam M, Ganesan AP, Chawla A, Djukanovic R, Ansel KM, Peters B, Rao A, Vijayanand P (2014) Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility. Nat Immunol 15(8):777–788. https://doi.org/10.1038/ni.2937
Finkelman FD, Shea-Donohue T, Morris SC, Gildea L, Strait R, Madden KB, Schopf L, Urban JF Jr (2004) Interleukin-4- and interleukin-13-mediated host protection against intestinal nematode parasites. Immunol Rev 201:139–155
Bao K, Carr T, Wu J, Barclay W, Jin J, Ciofani M, Reinhardt RL (2016) BATF modulates the Th2 locus control region and regulates CD4+ T cell fate during antihelminth immunity. J Immunol 197(11):4371–4381. https://doi.org/10.4049/jimmunol.1601371
Mohrs M, Shinkai K, Mohrs K, Locksley RM (2001) Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter. Immunity 15(2):303–311
Reinhardt RL, Liang HE, Bao K, Price AE, Mohrs M, Kelly BL, Locksley RM (2015) A novel model for IFN-gamma-mediated autoinflammatory syndromes. J Immunol 194(5):2358–2368. https://doi.org/10.4049/jimmunol.1401992
Acknowledgments
Thanks to the William Greenleaf lab for publishing this method and to the helpful discussions at the ATAC-seq users online Google Group forum: https://sites.google.com/site/atacseqpublic/. This research was funded in part by NIH grants RO1HL127461, RO1HL126600 (B.P.O.), and AI119004 (R.L.R).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Harmacek, L.D., Patel, P., Woolaver, R., Reinhardt, R.L., O’Connor, B.P. (2018). Library Preparation for ATAC-Sequencing of Mouse CD4+ T Cells Isolated from the Lung and Lymph Nodes After Helminth Infection. In: Reinhardt, R. (eds) Type 2 Immunity. Methods in Molecular Biology, vol 1799. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-7896-0_23
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7896-0_23
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-7895-3
Online ISBN: 978-1-4939-7896-0
eBook Packages: Springer Protocols