Abstract
BackgroundThe Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation.ResultsIn this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming.ConclusionsWe propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.
Highlights
The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes
We started our study by benchmarking the published DNAse Hi-C protocol developed by Ma et al [22]
We showed that the use of biotinylated adapters was not necessary and that biotin could be incorporated by applying a fill-in strategy
Summary
The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The coupling of the chromatin conformation capture technique with next-generation sequencing has resulted in the development of a simple and efficient Hi-C protocol, which enables the genome-wide chromatin architecture to be studied [1, 2]. Adjacent genomic segments interact considerably more frequently than distal or interchromosomal regions This dependence of chromatin contacts on genomic distance has been observed in all studied. Classical Hi-C protocols rely on restriction enzymes for fragmenting genomic DNA [1, 2] This fragmentation limits the theoretical resolution of Hi-C analysis by the restriction fragment length and results in nonuniform genomic coverage biased towards the regions flanking the Gridina et al Epigenetics & Chromatin (2021) 14:15 restriction enzyme recognition sites. For capture-Hi-C data, as well as for scaffolding or genotyping applications, high resolution and uniform coverage are desirable
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