An optimized 4C-seq protocol based on cistrome and epigenome data in the mouse RAW264.7 macrophage cell line

In this protocol, we describe the 4C-seq method in detail using RAW264.7 cells, a mouse macrophage cell line widely used to study acute and metabolic inflammation. We specifically outline how cistrome and epigenome data can be integrated into the primer designing step, critical for the entire protocol. Because intra-TAD chromatin loops are facilitated by transcription factors and coregulators, many of which are co-localized in open chromatin regions, the binding centers of those factors can be obtained by chromatin immunoprecipitation sequencing (ChIP-seq). The binding sites of these factors reflect the coherent loci of the chromatin loops and therefore can be used as references to improve the accuracy of the 4C primers. We specifically describe the 4C protocol using examples of recently identified Ccl2 enhancer and silencer as bait.