Multiplexed analysis of chromosome conformation at vastly improved sensitivity
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ABSTRACT: Current methods for analysing chromosome conformation in mammalian cells are either insensitive and low resolution or low throughput. Since the methods are expensive and relatively difficult to perform and analyse they are not widely used outside of specialised laboratories. Here we have re-designed the Capture-C method producing a new approach, called next generation (NG) Capture-C, which solves most of the current setbacks in analysing chromosome conformation. NG Capture-C produces unprecedented levels of sensitivity and reproducibility, which can be used to analyse any number of genetic loci and/or many samples in a single experiment. NG Capture is straightforward to perform, requiring only standard reagents and access to basic next generation sequencing platforms. The complete and detailed protocol presented here, with new publically available tools for library design and data analysis, will allow most laboratories to analyse chromatin conformation at levels of sensitivity and throughput that were previously impossible.
Project description:NG Capture-C provides high resolution chromatin conformation capture (3C) interaction maps. NG Capture-C was carried out in erythroid and hESC cells at the Hba, Hbb, Myc, and Slc25a37 locus to generate sequence libraries for analytical software testing.
Project description:Chromosome conformation capture (3C) provides an adaptable tool through which to study diverse biological questions. Currently, 3C techniques provide either low-resolution interaction profiles across the entire genome, e.g. HiC, or high-resolution interaction profiles at up to several hundred loci, e.g. NG Capture-C and 4C-seq. Generation of high-resolution, genome-wide interaction profiles can feasibly be achieved through efficiency improvements to current high-resolution methods. To this end we systematically tested and removed areas inefficiency in NG Capture-C to develop a new method Nuclear Capture-C, which provides a 300% increase in informative sequencing content. Using Nuclear Capture-C we target 8,026 erythroid promoters in triplicate, showing that this method can achieve high-resolution genome-wide 3C interaction profiles at scale.
Project description:Conformation capture-approaches like Hi-C can elucidate chromosome structure at a genome-wide scale. Hi-C datasets are large and require specialised software. Here, we present GENOVA: a user-friendly software package to analyse and visualise conformation capture data. GENOVA is an R-package that includes the most common Hi-C analyses, such as compartment and insulation score analysis. It can create annotated heatmaps to visualise the contact frequency at a specific locus and aggregate Hi-C signal over a user-specified genomic regions such as ChIP-seq data. Finally, our package supports output from the major mapping-pipelines. We demonstrate the capabilities of GENOVA by analysing Hi-C data from HAP1 cell lines in which the cohesin-subunits SA1 and SA2 were knocked out. We find that ΔSA1 cells gain intra-TAD interactions and increase compartmentalisation. ΔSA2 cells have longer loops and a less compartmentalised genome. These results suggest that cohesinSA1 forms longer loops, while cohesinSA2 plays a role in forming and maintaining intra-TAD interactions. The differences in loop-forming activity affect whole chromosome organisation consistent with a model where loops and compartments counterbalance each other. We show that GENOVA is an easy to use R-package, that allows researchers to explore Hi-C data in great detail.
Project description:ING1b and GADD45a are nuclear proteins involved in the regulation of cell growth, apoptosis and DNA repair. We previously found that ING1b is essential to target GADD45a-mediated active DNA-demethylation via TET1 to specific loci. In order to study the physical interaction of distant GADD45a and ING1 bound regions, we performed multiplexed NG Capture-C chromatin conformation capture assay in wildtype and knockout mouse embryonic fibroblasts.
Project description:We present HiChew (High efficient chromatin conformation capture with post-enrichment), a novel method for analyzing three-dimensional chromatin organization in single cells and low-input samples. HiChew combines efficient sticky-end ligation with post-PCR enrichment using methylation-based selection, addressing key limitations of existing chromatin conformation capture methods. The method achieves significantly higher valid pair ratios (approximately 50%) compared to unenriched methods (8%) while maintaining high capture sensitivity. For single-cell applications, snHiChew demonstrates superior performance with 45-50% valid pair ratios and the ability to generate up to 7.3 million unique valid contacts per cell. This enhanced efficiency enables high-resolution mapping of chromatin interactions at 5-10 kb resolution with 70-80% bin coverage. We validate HiChew's accuracy through comparative analyses with conventional Hi-C data, showing high correlations in compartment scores, topologically associating domains (TADs), and loop detection. The method's scalability and cost-effectiveness make it particularly suitable for large-scale single-cell chromatin conformation studies. HiChew represents a significant advancement in chromatin architecture analysis, offering improved efficiency without compromising data quality or sensitivity.
Project description:The spatiotemporal chromatin reorganization during hematopoietic differentiation has not been well characterized, which partly limited by the large amounts of starting cells for the current high-throughput chromatin conformation capture approaches. Here, we introduce a low-input Hi-C method, tagHi-C, to capture the chromatin structures in hundreds of cells. We proved tagHi-C was a feasible and reproducible approach
Project description:The spatiotemporal chromatin reorganization during hematopoietic differentiation has not been well characterized, which partly limited by the large amounts of starting cells for the current high-throughput chromatin conformation capture approaches. Here, we introduce a low-input Hi-C method, tagHi-C, to capture the chromatin structures in hundreds of cells. We proved tagHi-C was a feasible and reproducible approach.
Project description:For more than a decade, microarrays have been a powerful and widely used tool to explore the transcriptome of biological systems. However, the amount of biological material from cell sorting or laser capture microdissection is much too small to perform microarray studies. To address this issue, RNA amplification methods have been developed to generate sufficient targets from picogram amounts of total RNA to perform microarray hybridisation. In this study, four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. The quantitative and qualitative performances of the methods were assessed. Microarrays were hybridised with the amplified targets and the amplification protocols were compared with respect to the quality of expression profiles, reproducibility within a concentration range of input RNA, and sensitivity. Four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. For each protocol, one RNA amplification was performed from 250 pg, and one from 500 pg of human universal RNA by two operators in two independent laboratories and compared to the amplified aRNA obtained from 2 µg and 100 ng RNA inputs following the standard protocol proposed by Affymetrix. A negative control (amplification without total RNA) and a positive control (if available) were included in each experimental batch. Samples indicating 50, 100, and 1000 pg RNA inputs correspond to 3 additional quantities of total RNA used to synthesise the cDNA target using the nugen protocol for comparison (250, 500 pg + 50, 100, 1000 pg).
Project description:We developed SLIC-CAGE (Super-Low Input Carrier-CAGE) approach to capture 5'end of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity compared to existing CAGE methods is achieved by specially designed, selectively degradable carrier RNA. We tested SLIC-CAGE on Saccharomyces cerevisiae (BY4741 strain) and produced libraries from 1-100 ng of total cellular RNA. We also produced S. cerevisiae nAnT-iCAGE libraries as the current gold-standard CAGE libraries using the recommended 5 micrograms of total cellular RNA to assess the quality of SLIC-CAGE libraries produces with up to 1000-fold less material. We provide a direct comparison between SLIC-CAGE and the latest nanoCAGE protocol (libraries created using S. cerevisiae total RNA) and show that SLIC-CAGE produces unbiased libraries of higher complexity and quality than nanoCAGE. Finally, we provide SLIC-CAGE libraries on mouse embryonic stem cells (E14) using 5-100 ng of total cellular RNA as starting material.