Chromosome Conformation Capture (3C) and 3C-based technologies are constantly evolving in order to probe nuclear organization with higher depth and resolution. One such method is 4C-technology that allows the investigation of the nuclear environment of a locus of choice. The use of Illumina next generation sequencing as a detection platform for the analysis of 4C data has further improved the sensitivity and resolution of this method. Here we provide a step-by-step protocol for 4C-seq, describing the procedure from the initial template preparation until the final data analysis, interchanged with background information and considerations.