Schematic representation of 5C. (A) A 3C library is generated by conventional 3C and then converted into a 5C library by annealing and ligating 5C oligonucleotides in a multiplex setting. 5C libraries are then analyzed on a microarray or by quantitative sequencing. (B) 5C primer design. Forward 5C primers anneal to the sense strand of the 3′-end of restriction fragments and include half of the selected restriction site. All forward primers feature a common 5′-end tail containing the T7 promoter sequence. Reverse 5C primers anneal to the antisense strand of the 3′-end of restriction fragments, including half of the restriction site. All reverse primers contain a common 3′-end tail featuring the complementary T3 sequence (T3c) and are phosphorylated at the 5′-end. 5C forward and reverse primers anneal to the same strand of head-to-head ligation products present in the 3C library.
