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