title: From cells to chromatin: capturing snapshots of genome organization with 5C technology.
abstract: In eukaryotes, genome organization can be observed on many levels and at different scales. This organization is important not only to reduce chromosome length but also for the proper execution of various biological processes. High-resolution mapping of spatial chromatin structure was made possible by the development of the chromosome conformation capture (3C) technique. 3C uses chemical cross-linking followed by proximity-based ligation of fragmented DNA to capture frequently interacting chromatin segments in cell populations. Several 3C-related methods capable of higher chromosome conformation mapping throughput were reported afterwards. These techniques include the 3C-carbon copy (5C) approach, which offers the advantage of being highly quantitative and reproducible. We provide here an updated reference protocol for the production of 5C libraries analyzed by next-generation sequencing or onto microarrays. A procedure used to verify that 3C library templates bear the high quality required to produce superior 5C libraries is also described. We believe that this detailed protocol will help guide researchers in probing spatial genome organization and its role in various biological processes.
authors.affiliation: Department of Biochemistry and Goodman Cancer Research Center, McGill University, 3655 Promenade Sir-William-Osler, Room 815A, Montréal, Québec, Canada H3G1Y6. maria.ferraiuolo@mail.mcgill.ca
authors.forename: Maria A
authors.initials: MA
authors.lastname: Ferraiuolo
authors.forename: Amartya
authors.initials: A
authors.lastname: Sanyal
authors.forename: Natalia
authors.initials: N
authors.lastname: Naumova
authors.forename: Job
authors.initials: J
authors.lastname: Dekker
authors.forename: Josée
authors.initials: J
authors.lastname: Dostie
mesh_terms: D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D005557:Formaldehyde / Q000737:chemistry; D015723:Gene Library; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D016707:Tissue Fixation; D017186:Titrimetry
chemical_list: D002843:Chromatin; D003432:Cross-Linking Reagents; D017931:DNA Primers; D005557:Formaldehyde; D004247:DNA