Project description:Regulatory DNA elements can control expression of distant genes via physical interactions. Here, we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using Chromosome Conformation Capture combined with high-throughput sequencing (4C-seq) data. Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation. A high resolution 4C-seq protocol involving two restriction digests and a revised analysis pipeline was applied to several viewpoints in four genomic loci (the well-characterized alpha-globin and beta-globin loci, and the novel Oct4 and Satb1 loci), allowing robust detection of physical interactions between regulatory DNA elements.
Project description:Hi-C technique is widely used to study 3-dimensional chromatin architecture and assemble genomes. Conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in non-uniform genomic coverage. Using sequence-agnostic restriction enzymes such as DNAse I could overcome this limitation. Here we compared different DNAse Hi-C protocols and identified several critical steps which significantly impact protocol efficiency. We proposed a new robust protocol for preparation of DNAse Hi-C libraries, supplemented with experimental controls and computational pipeline for evaluation of libraries quality and data analysis.
