4C Analysis of 3C, ChIP-Loop, and Control Libraries.
The 4C detection method starts with a 3C, ChIP-loop, or control library and then uses inverse polymerase chain reaction (PCR) to amplify all restriction fragments that are ligated to a single restriction fragment of interest. As a result, a genome-wide interaction profile of the restriction fragment of interest is obtained. Before starting a 4C experiment, first assess the quality of the 3C, ChIP-loop, or control library that will be studied by performing semiquantitative PCR with the library as template. This procedure ascertains that ligation products can be readily detected and that nearby restriction fragments become ligated more frequently than more distant fragments. Choice of a restriction enzyme for 4C analysis and determination of interaction frequencies using 4C are described in detail.