Chromosome Conformation Capture with Deep Sequencing to Study the Roles of the Structural Maintenance of Chromosomes Complex In Vivo
Recent applications of chromosome conformation capture with deep sequencing (Hi-C and other C techniques) has enabled high-throughput investigations and driven major advances in understanding chromosome organization in bacteria and eukaryotes. C techniques reveal systematically the identities of interacting DNA and the frequency of each interaction in vivo. Beyond a bird’s-eye view survey of the global chromosome architecture, C techniques together with genetic perturbation have proven to be powerful in understanding factors that shape chromosome architectures. The structural maintenance of chromosomes (SMC) proteins play major roles in organizing the chromosomes from bacteria to humans, and C techniques have contributed to understanding their mechanism and impact on genome organization in a cellular context. Here, I describe a Hi-C protocol, a variant of C techniques, to construct genome-wide DNA contact maps for bacteria. This protocol is optimized for the gram-negative bacterium Caulobacter crescentus, but it can be readily adapted for any bacterial species of interest.
Key wordsChromosome conformation capture Hi-C Deep sequencing Chromosome organization Structural maintenance of chromosomes SMC Caulobacter crescentus
This research is supported by a Royal Society University Research Fellowship (UF140053 and RG150448) and a BBSRC grant (BB/P018165/1) to T.B.K.L. The Hi-C technique was first optimized for Caulobacter crescentus when the author was a postdoctoral fellow in the lab of Prof. Michael Laub. The author thanks Prof. Michael Laub and Dr. Mark Umbarger for support and advice during the optimization of this protocol.
- 7.Badrinarayanan A, Le TBK, Laub MT (2015) Bacterial chromosome organization and segregation. Annu Rev Cell Dev Biol 31:171–199. https://doi.org/10.1146/annurev-cellbio-100814-125211 CrossRefPubMedPubMedCentralGoogle Scholar
- 28.Viollier PH, Thanbichler M, McGrath PT et al (2004) Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. Proc Natl Acad Sci U S A 101:9257–9262. https://doi.org/10.1073/pnas.0402606101 CrossRefPubMedPubMedCentralGoogle Scholar
- 29.Schrader JM, Shapiro L (2015) Synchronization of Caulobacter crescentus for investigation of the bacterial cell cycle. J Vis Exp. https://doi.org/10.3791/52633
- 36.van Berkum NL, Lieberman-Aiden E, Williams L et al (2010) Hi-C: a method to study the three-dimensional architecture of genomes. J Vis Exp. https://doi.org/10.3791/1869