Robust detection of chromosomal contacts from small cell numbers using low-input Capture-C
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE99257 | GEO | 2017/11/20
SECONDARY ACCESSION(S): PRJNA387734
REPOSITORIES: GEO
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2014-01-15 | E-GEOD-52281 | biostudies-arrayexpress
Project description:Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin architecture in rare cell populations. We present a new low-input Capture-C approach that can generate high-quality 3C interaction profiles from 10 000-20 000 cells, depending on the resolution used for analysis. We also present a PCR-free, sequencing-free 3C technique based on NanoString technology called C-String. By comparing C-String and Capture-C interaction profiles we show that the latter are not skewed by PCR amplification. Furthermore, we demonstrate that chromatin interactions detected by Capture-C do not depend on the degree of cross-linking by performing experiments with varying formaldehyde concentrations.
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Project description:Though important for gene regulation most studies of genome organisation use either fluorescence in situ hybridisation (FISH) or chromosome conformation capture (3C) methods. FISH directly visualises the spatial relationship of sequences, but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde crosslinking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organisation, but this has not been tested extensively. We investigate the murine HoxD locus with 5C and FISH in different developmental and activity states, and in the presence or absence of epigenetic regulators. We identify situations where the two datasets are concordant, but find other conditions where chromatin topographies extrapolated from 5C or FISH data are not compatible. We conclude that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart – influenced by nuclear environment and chromatin composition. We conclude that results obtained at high-resolution with either 3C methods or by FISH alone must be interpreted with caution and that conclusions about genome organisation should be validated by independent methods. 5C oligonucleotides were designed around EcoRI restriction sites following an alternative scheme
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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.
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