ChIP-on-chip protocol for genome-wide analysis of transcription factor binding in Drosophila melanogaster embryos.
This protocol describes a method to detect in vivo associations between proteins and DNA in developing Drosophila embryos. It combines formaldehyde crosslinking and immunoprecipitation of protein-bound sequences with genome-wide analysis using microarrays. After crosslinking, nuclei are enriched using differential centrifugation and the chromatin is sheared by sonication. Antibodies specifically recognizing wild-type protein or, alternatively, a genetically encoded epitope tag are used to enrich for specifically bound DNA sequences. After purification and polymerase chain reaction-based amplification, the samples are fluorescently labeled and hybridized to genomic tiling microarrays. This protocol has been successfully used to study different tissue-specific transcription factors, and is generally applicable to in vivo analysis of any DNA-binding proteins in Drosophila embryos. The full protocol, including the collection of embryos and the collection of raw microarray data, can be completed within 10 days.
Sandmann
Thomas
T
EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Jakobsen
Janus S
JS
Furlong
Eileen E M
EE
D000818:Animals; D001665:Binding Sites; D003627:Data Interpretation, Statistical; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D049750:Genome, Insect; D023281:Genomics / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012107:Research Design; D014157:Transcription Factors / Q000378:metabolism*
D014157:Transcription Factors