Skip to main content
SpringerLink
Log in

Dual Cross-Linking Chromatin Immunoprecipitation Protocol for Next-Generation Sequencing (ChIPseq) in Macrophages

  • Protocol
  • First Online:
Lipid-Activated Nuclear Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1951))

Abstract

Macrophages arise from distinct progenitor cell populations throughout development and are one of the most diverse cell types, capable of performing discrete functions, undergoing distinct modes of activation, and infiltrating or residing in numerous niches in the body. In adapting to their environments, macrophages display high levels of plasticity which is associated with profound epigenomic and transcriptional changes. Understanding these changes has been greatly facilitated by the next-generation sequencing (NGS)-based approaches such as RNAseq and chromatin immunoprecipitation (ChIP)seq. Despite the recent advances, obtaining quality ChIPseq data in macrophages for endogenous factors and especially coregulators recruited to DNA indirectly has proved to be extremely challenging. Here, we describe a dual crosslinking protocol for ChIPseq in macrophages that we developed for difficult-to-ChIP transcription factors, coregulators, and their posttranslational modifications. Further, we provide guidance on crucial optimization steps throughout this protocol. Although our experience has been predominantly in murine and human macrophages, we believe our protocols can be modified and optimized to study signal-induced epigenomic changes in any cell type of choice.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wynn TA, Chawla A, Pollard JW (2013) Macrophage biology in development, homeostasis and disease. Nature 496(7446):445–455. https://doi.org/10.1038/nature12034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Xue J, Schmidt SV, Sander J, Draffehn A, Krebs W, Quester I, De Nardo D, Gohel TD, Emde M, Schmidleithner L, Ganesan H, Nino-Castro A, Mallmann MR, Labzin L, Theis H, Kraut M, Beyer M, Latz E, Freeman TC, Ulas T, Schultze JL (2014) Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40(2):274–288. https://doi.org/10.1016/j.immuni.2014.01.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, Giustetto M, Ferreira TA, Guiducci E, Dumas L, Ragozzino D, Gross CT (2011) Synaptic pruning by microglia is necessary for Normal brain development. Science 333(6048):1456–1458. https://doi.org/10.1126/science.1202529

    Article  CAS  PubMed  Google Scholar 

  4. Nathan C, Ding A (2010) Nonresolving inflammation. Cell 140(6):871–882. https://doi.org/10.1016/j.cell.2010.02.029

    Article  CAS  PubMed  Google Scholar 

  5. Ho JW, Bishop E, Karchenko PV, Negre N, White KP, Park PJ (2011) ChIP-chip versus ChIP-seq: lessons for experimental design and data analysis. BMC Genomics 12:134. https://doi.org/10.1186/1471-2164-12-134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Buermans HP, den Dunnen JT (2014) Next generation sequencing technology: advances and applications. Biochim Biophys Acta 1842(10):1932–1941. https://doi.org/10.1016/j.bbadis.2014.06.015

    Article  CAS  PubMed  Google Scholar 

  7. Fonseca GJ, Seidman JS, Glass CK (2016) Genome-wide approaches to defining macrophage identity and function. Microbiol Spectr 4(5). https://doi.org/10.1128/microbiolspec.MCHD-0039-2016

  8. Goi C, Little P, Xie C (2013) Cell-type and transcription factor specific enrichment of transcriptional cofactor motifs in ENCODE ChIP-seq data. BMC Genomics 14(Suppl 5):S2. https://doi.org/10.1186/1471-2164-14-s5-s2

    Article  PubMed  PubMed Central  Google Scholar 

  9. Schmidt SV, Krebs W, Ulas T, Xue J, Baßler K, Günther P, Hardt A-L, Schultze H, Sander J, Klee K, Theis H, Kraut M, Beyer M, Schultze JL (2016) The transcriptional regulator network of human inflammatory macrophages is defined by open chromatin. Cell Res 26:151. https://doi.org/10.1038/cr.2016.1 http://www.nature.com/articles/cr20161—supplementary-information

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rollins DA, Coppo M, Rogatsky I (2015) Minireview: nuclear receptor coregulators of the p160 family: insights into inflammation and metabolism. Mol Endocrinol 29(4):502–517. https://doi.org/10.1210/me.2015-1005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nowak DE, Tian B, Brasier AR (2005) Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation. BioTechniques 39(5):715–725

    Article  CAS  Google Scholar 

  12. Rollins DA, Kharlyngdoh JB, Coppo M, Tharmalingam B, Mimouna S, Guo Z, Sacta MA, Pufall MA, Fisher RP, Hu X, Chinenov Y, Rogatsky I (2017) Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages. Nat Commun 8(1):1739. https://doi.org/10.1038/s41467-017-01569-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sacta MA, Tharmalingam B, Coppo M, Rollins DA, Deochand DK, Benjamin B, Yu L, Zhang B, Hu X, Li R, Chinenov Y,Rogatsky I (2018) Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages. eLife 7:e34864 https://doi.org/10.7554/eLife.34864

    Article  Google Scholar 

  14. Muir P, Li S, Lou S, Wang D, Spakowicz DJ, Salichos L, Zhang J, Weinstock GM, Isaacs F, Rozowsky J, Gerstein M (2016) The real cost of sequencing: scaling computation to keep pace with data generation. Genome Biol 17:53. https://doi.org/10.1186/s13059-016-0917-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nakato R, Shirahige K (2017) Recent advances in ChIP-seq analysis: from quality management to whole-genome annotation. Brief Bioinform 18(2):279–290. https://doi.org/10.1093/bib/bbw023

    Article  CAS  PubMed  Google Scholar 

  16. Kidder BL, Hu G, Zhao K (2011) ChIP-Seq: technical considerations for obtaining high-quality data. Nat Immunol 12(10):918–922. https://doi.org/10.1038/ni.2117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgment

The authors are grateful to the support of the NIH NIAMS T32 AR007281 to DAR, the grants to IR from the NIH R01DK099087, the Rheumatology Research Foundation, the DOD CDMRP PR130049, and the Hospital for Special Surgery David Rosensweig Genomics Center. We thank Dr. Y Chinenov (HSS) for critical comments on the manuscript.

Author information

Authors and Affiliations

  1. Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA

    David A. Rollins & Inez Rogatsky

  2. The David Rosensweig Genomics Center, Hospital for Special Surgery Research Institute, New York, NY, USA

    David A. Rollins & Inez Rogatsky

  3. Mayo Clinic School of Medicine M.D. Program, Rochester, MN, USA

    David A. Rollins

Authors
  1. David A. Rollins
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Inez Rogatsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Inez Rogatsky .

Editor information

Editors and Affiliations

  1. Division of Medicine, Centre for Cardiometabolic Medicine, University College London, London, UK

    Matthew C. Gage

  2. Division of Medicine, Centre for Cardiometabolic Medicine, University College London, London, UK

    Inés Pineda-Torra

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Rollins, D.A., Rogatsky, I. (2019). Dual Cross-Linking Chromatin Immunoprecipitation Protocol for Next-Generation Sequencing (ChIPseq) in Macrophages. In: Gage, M., Pineda-Torra, I. (eds) Lipid-Activated Nuclear Receptors. Methods in Molecular Biology, vol 1951. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9130-3_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9130-3_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9129-7

  • Online ISBN: 978-1-4939-9130-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation