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Methods Mol Biol. 2012;809:3-26. doi: 10.1007/978-1-61779-376-9_1.

Genome-wide in vivo cross-linking of sequence-specific transcription factors.

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  • 1Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

Abstract

Immunoprecipitation of cross-linked chromatin in combination with microarrays (ChIP-chip) or ultra high-throughput sequencing (ChIP-seq) is widely used to map genome-wide in vivo transcription factor binding. Both methods employ initial steps of in vivo cross-linking, chromatin isolation, DNA fragmentation, and immunoprecipitation. For ChIP-chip, the immunoprecipitated DNA samples are then amplified, labeled, and hybridized to DNA microarrays. For ChIP-seq, the immunoprecipitated DNA is prepared for a sequencing library, and then the library DNA fragments are sequenced using ultra high-throughput sequencing platform. The protocols described here have been developed for ChIP-chip and ChIP-seq analysis of sequence-specific transcription factor binding in Drosophila embryos. A series of controls establish that these protocols have high sensitivity and reproducibility and provide a quantitative measure of relative transcription factor occupancy. The quantitative nature of the assay is important because regulatory transcription factors bind to highly overlapping sets of thousands of genomic regions and the unique regulatory specificity of each factor is determined by relative moderate differences in occupancy between factors at commonly bound regions.

PMID:
22113265
[PubMed - indexed for MEDLINE]
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