Project description:Chromosome conformation capture (3C) and derivative (4C, 5C and Hi-C) methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. We previously described an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing "in-nucleus ligation" and the standard "in-solution ligation". The results show that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. Thus in-nucleus ligation not only simplifies the experimental procedures, but also produces higher quality data with benefits for the entire range of 3C applications. We created Hi-C libraries by two different methods, in-solution ligation and in-nucleus ligation, from two biological replicates each of mouse foetal liver cells (mouse-1 and mouse-2) and human ES cells (human-1 and human-2) or the mixture of these two species. We also sequenced a random ligation library prepared by reversal of the cross-links and purification of the DNA prior to ligation.
Project description:Chromosome conformation capture (3C) and derivative (4C, 5C and Hi-C) methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. We previously described an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing "in-nucleus ligation" and the standard "in-solution ligation". The results show that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. Thus in-nucleus ligation not only simplifies the experimental procedures, but also produces higher quality data with benefits for the entire range of 3C applications.
Project description:Chemical cross-linking and high-throughput sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no such interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of chromatin folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to ten-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes. Hi-C experiments where ligation is performed on beads (tethered) on male Drosophila salivary glands from three independent biological replicates. Also one Hi-C experiment where the ligation is performed in solution (conventional).
Project description:CD34+ heamatopoietic stem cells were isolated from the bone marrow of two healthy donors undergoing total hip replacement. Promoter capture Hi-C (PCHi-C) was performed on these cells using the protocol according to Mifsud et al. 2015, with the exception that ligation was performed in situ, and a slightly modified bait capture set was used. Bait positions in hg19 are included as an additional file.
Project description:Rapid advances in high-throughput DNA sequencing technologies are accelerating the pace of research into personalized medicine. While methods for variant discovery and genotyping from whole genome sequencing (WGS) datasets have been well established, linking variants together into a single haplotype remains a challenge. An understanding of complete haplotypes of an individual will help clarify the consequences of inheriting multiple alleles in combination, identify novel disease associations, and augment studies of gene regulation. Although numerous methods have been developed to reconstruct haplotypes from WGS data, chromosome-span haplotypes at high resolution have been difficult to obtain. Here we present a novel method to accurately reconstruct chromosome-span haplotypes from proximity-ligation and DNA shotgun sequencing. We demonstrate the utility of this approach in producing high-resolution chromosome-span haplotype phasing in mouse and human. While proximity-ligation based methods were originally designed to investigate spatial organization of the genome, our results lend support for their use as a general tool for haplotyping in the future. Hi-C experiments in two replicates of Human GM12878 Lymphoblastoid cells and two replicates of F123 mouse ES cells (4 total samples)
Project description:In this project, we use a simple digestion-ligation-only Hi-C (DLO Hi-C) technology, for whole-genome chromosome conformation capture, analysis of genes expression changes and transcriptional regulatory elements involved in BMMSC differentiation. Rely on GWAS data and eQTL analysis, our findings confirmed some previously reported genes that have an effect on OP, such as TIMP-2, MMP-2 and DAAM2, TMEM241 was less reported. Furthermore, our work provide a theoretical basis for the development of OP.