Abstract
Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip and ChIP-sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here, we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms.
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Abbreviations
- bp:
-
Base pair(s)
- BSA:
-
Bovine serum albumin
- cDNA:
-
Complementary DNA
- ChIP:
-
Chromatin immunoprecipitation
- CRM:
-
cis-regulatory module
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- PBS:
-
Phosphate-buffered saline
- PCR:
-
Polymerase chain reaction
- qPCR:
-
Quantitative PCR
- wt:
-
Wild type
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Acknowledgments
We are indebted to Prof. Fiorenza De Bernardi (University of Milan, Italy) for the immunofluorescence protocol. This work was supported by grant NIH/NIGMS GM100466 along with supplemental funding from the American Recovery and Reinvestment Act award R01HD050704-05S1 to ADG and by grants from the American Cancer Society (RSG-08-042-01-DDC) and the Charles A. Frueauff Foundation to YN. HA was supported in part by a postdoctoral fellowship from the Japan Society for the Promotion of Science (JSPS).
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The authors declare no conflict of interest.
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Hitoshi Aihara and Lavanya Katikala contributed equally to this work (as first authors).
Anna Di Gregorio and Yutaka Nibu contributed equally to this work (as senior authors).
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Aihara, H., Katikala, L., Zeller, R.W. et al. Optimization of a Method for Chromatin Immunoprecipitation Assays in the Marine Invertebrate Chordate Ciona . Mar Biotechnol 15, 520–525 (2013). https://doi.org/10.1007/s10126-013-9504-5
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DOI: https://doi.org/10.1007/s10126-013-9504-5