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Erythropoiesis pp 229-236 | Cite as

Chromatin Immunoprecipitation (ChIP) with Erythroid Samples

  • Ivan Krivega
  • Ann Dean
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1698)

Abstract

Chromatin immunoprecipitation (ChIP) allows determination of the locations to which a select protein is bound in chromatin. Chemical crosslinking of DNA and protein with bi-functional reagents such as formaldehyde and precipitation of the protein with a specific antibody permit PCR amplification (ChIP) or sequencing (ChIP-seq) to identify the bound sites. Here, we present methodology for these approaches that are widely applicable to erythroid cell lines, progenitor cells, and tissues.

Key words

Chromatin DNA-binding protein ChIP ChIP-seq Crosslinking Antibody Immunoprecipitation 

Notes

Acknowledgments

This work was supported by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK015508 to AD).

References

  1. 1.
    Hoffman EA, Frey BL, Smith LM, Auble DT (2015) Formaldehyde crosslinking: a tool for the study of chromatin complexes. J Biol Chem 290:26404–26411CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    O’Neill LP, Turner BM (2003) Immunoprecipitation of native chromatin: NChIP. Methods 31:76–82CrossRefPubMedGoogle Scholar
  3. 3.
    Baranello L, Kouzine F, Sanford S, Levens D (2016) ChIP bias as a function of cross-linking time. Chromosom Res 24:175–181CrossRefGoogle Scholar
  4. 4.
    Krivega I, Dale RK, Dean A (2014) Role of LDB1 in the transition from chromatin looping to transcription activation. Genes Dev 28:1278–1290CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Krivega I, Byrnes C, de Vasconcellos JF, Lee YT, Kaushal M, Dean A, Miller JL (2015) Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/gamma globin looping. Blood 126:665–672CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar
  7. 7.
    Zeng PY, Vakoc CR, Chen ZC, Blobel GA, Berger SL (2006) In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. Biotechniques 41:694, 696, 698CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Laboratory of Cellular and Developmental BiologyNational Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of HealthBethesdaUSA

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