Sister chromatid-sensitive Hi-C to map the conformation of replicated genomes

Chromosome conformation capture (Hi-C) techniques map the 3D organization of entire genomes. How sister chromatids fold in replicated chromosomes, however, cannot be determined with conventional Hi-C because of the identical DNA sequences of sister chromatids. Here, we present a protocol for sister chromatid-sensitive Hi-C (scsHi-C) that enables the distinction of DNA contacts within individual sister chromatids (cis sister contacts) from those between sister chromatids (trans sister contacts), thereby allowing investigation of the organization of replicated genomes. scsHi-C is based on live-cell labeling of nascent DNA by the synthetic nucleoside 4-thio-thymidine (4sT), which incorporates into a distinct DNA strand on each sister chromatid because of semi-conservative DNA replication. After purification of genomic DNA and in situ Hi-C library preparation, 4sT is chemically converted into 5-methyl-cytosine in the presence of OsO₄/NH₄Cl to introduce T-to-C signature point mutations on 4sT-labeled DNA. The Hi-C library is then sequenced, and ligated fragments are assigned to sister chromatids on the basis of strand orientation and the presence of signature mutations. The ensemble of scsHi-C contacts thereby represents genome-wide contact probabilities within and across sister chromatids. scsHi-C can be completed in 2 weeks, has been successfully applied in HeLa cells and can potentially be established for any cell type that allows proper cell cycle synchronization and incorporation of sufficient amounts of 4sT. The genome-wide maps of replicated chromosomes detected by scsHi-C enable investigation of the molecular mechanisms shaping sister chromatid topologies and the relevance of sister chromatid conformation in crucial processes like DNA repair, mitotic chromosome formation and potentially other biological processes.