Abstract
Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples (A), cross-linking proteins to DNA (B), chromatin isolation and fragmentation by sonication (C), sonication test (D), immunoprecipitation with antibodies against the protein or the histone mark of interest (E), DNA recovery (E), identification of factor-associated DNA sequences by PCR or sequencing (F). The protocol described here can readily be used for ChIP-seq and ChIP-qPCR experiments. The entire procedure, describing experimental setup conditions to optimize assays in intact Drosophila tissues, can be completed within four days.
Keywords: ChIP, Drosophila, Embryo, Imaginal disc, Epigenetic mark, Transcription factor
Background
Despite the fact that immortalized cultured cells are extensively used to study the chromatin landscape of various cell types, valuable methods for probing interaction in vivo, under physiological conditions, are necessary to perform temporal or spatial comparative analysis of transcription factor and histone modification maps between different stages of Drosophila development or between different tissues. Here we present a detailed ChIP protocol that has been optimized to work on whole Drosophila embryos and larval imaginal discs, highlighting critical experimental parameters.
Materials and Reagents
Equipment
Procedure
IMAGINAL DISCS:
Note: All the following steps are done on ice or at 4 °C unless otherwise specified.
Data analysis
Notes
Recipes
Note: The solutions described below should not be stored for more than 1 week at 4 °C.
Acknowledgments
We thank all the authors of the article from which this protocol was adapted from (Loubiere et al., 2016). We thank Frédéric Bienvenu (Institute of Functional Genomics, Montpellier, France) for his technical support. We thank Filippo Ciabrelli for discussions and help in writing this manuscript.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Apologies,I can see you have used ~50 mg of wet tissue/ 1000 embryos per ChIP but would still be interested to what results you generally got from the Qubit initially after sonication and then once again after IP?Regards,Tom