Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip.
Chromosome conformation capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide coassociations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced chromosome conformation capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin coassociations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.
Laboratory of Chromatin and Cell Biology, Institute of Human Genetics, Montpellier, France.
Tom
T
Sexton
Sreenivasulu
S
Kurukuti
Jennifer A
JA
Mitchell
David
D
Umlauf
Takashi
T
Nagano
Peter
P
Fraser
D019921:Biotinylation; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D002875:Chromosomes / Q000737:chemistry* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D057890:Epigenomics / Q000379:methods*; D009690:Nucleic Acid Conformation
D002843:Chromatin; D017931:DNA Primers