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EpiTect Hi-C Kit

For high-resolution mapping of chromatin folding, high-quality assembly of genome sequences, haplotype phasing and identifying chromosomal rearrangements

  • All-inclusive kit – quality-controlled reagents for generating Hi-C NGS libraries
  • Included Illumina adapters with sequence bar codes for multiplex sequencing
  • Fully tested, robust and fast protocol – Hi-C library generation in <2 days
  • Low sample input requirement – libraries generated from just 250,000 cells
  • Data analysis pipeline based on open-source tools

Hi-C was originally conceived as a powerful technique for genome-wide chromosome conformation capture, enabling the characterization of chromatin folding at kb resolution. However, the technology also has other important applications. For example, Hi-C is used for generating highly contiguous genome assemblies, with few and very long scaffolds, from organisms without a known reference genome. In addition, Hi-C is also very useful for haplotype phasing and detection of chromosomal rearrangements.

The EpiTect Hi-C Kit offers a robust, yet simple and fast, protocol with low cell input requirements that enables generation of high-quality Hi-C Illumina NGS libraries from cross-linked cells in less than 2 days.

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Cat No./ID: 59971
EpiTect Hi-C Kit (6)
Reagents for generating Hi-C NGS libraries for up to 6 samples
The EpiTect Hi-C Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

Downstream applications of Hi-C sequencing data
Originally, the Hi-C method was developed to understand the relationship between chromosome conformation and gene expression. However, in recent years, Hi-C has been used successfully for additional applications such as de novo assembly of genomes, haplotype phasing and detection of chromosomal rearrangements.
Cis/Trans ratio
In the nucleus, chromosomes are partitioned in territories, where individual chromosomes are physically separated in space. For this reason, DNA contacts typically occur at higher frequency within chromosomes (cis) than between chromosomes (trans). This property of genome organization can be exploited as a useful proxy for evaluating the quality of Hi-C data. Noise from random background ligation (e.g., due to ruptured nuclei) will affect both cis and trans interactions similarly and result in a lower ratio between cis and trans interactions. Cis/trans ratios of 40–60% are considered to represent high quality Hi-C experiments (Lajoi et al. Methods, 2015, 72:65–75). The EpiTect Hi-C Kit generates much higher cis/trans ratios (on average 75%), which demonstrates that the protocol successfully maintains nuclear integrity until the Hi-C ligation event.
No strand orientation bias with the EpiTect Hi-C Kit

Hi-C proximity ligation of chromatin generates interaction pairs in the form of DNA chimeras. When valid Hi-C interactions pairs (DNA chimeras) are paired-end sequenced, read 1 (blue arrow) and read 2 (red arrow) will map to 2 different restriction fragments (A: blue line and red line). Hi-C chimeras can be divided into 4 classes, distinguished by the strand orientation of read pairs as shown in A. If the chimeras are a result of random proximity ligation of chromatin, then 25% of each class of chimera is expected. The absence of strand bias observed with EpiTect Hi-C NGS libraries, as shown in B, is further evidence of the high performance of the EpiTect Hi-C Kit.

Levels of chromatin organization
Genomes are organized into spatial hierarchies of increasing resolution, beginning with chromosomal territories and extending down to chromosomal compartments, topologically associating domains (TADs) and chromatin loops.
Percentage of paired reads deriving from a single restriction fragment
The proportion of invalid read pairs deriving from a single restriction fragment is also a good indicator of Hi-C library quality. This type of uninformative data derives from restriction fragments that either failed to ligate (dangling ends) or ligated with itself forming a circle (self-circle). A high-quality Hi‐C library for mammalian genomes typically has <1–4% unligated, dangling ends and <1–2% self-ligated circles – criteria that are reproducibly achieved with the EpiTect Hi-C Kit.
Percentage of long-range cis interactions
One of the most crucial metrics for assessing Hi-C library quality is the percentage of long-range cis (intra-chromosomal) contacts. Contacts within the same chromosome are considered long-range when the interaction is between two loci >20 kb apart. According to Rao et al. (Cell, 2014, 159:1665–1680), Hi-C libraries can be considered successful with >40% long-range interactions and excellent with >50%. The EpiTect Hi-C Kit exceeds these criteria by generating libraries with >60% long-range interactions.
Percentage of Hi-C events
Proximity ligation of chromatin in Hi-C generates interaction pairs (Hi-C events) in the form of DNA chimeras. When valid Hi-C interactions pairs (DNA chimeras) are paired-end sequenced, read 1 and read 2 will map to two different restriction fragments in the genome. According to guidelines by Rao et al. (Cell, 2014, 159:1665–1680), if <80% of valid read pairs come from Hi-C events, the library is likely the result of failed restriction, fill-in or ligation steps. As such, these Hi-C libraries are not considered suitable candidates for deeper sequencing. This data shows that EpiTect Hi-C libraries generate valid reads that are almost entirely made up of Hi-C events.
EpiTect Hi-C workflow – day 1

Day 1 of the EpiTect Hi-C workflow.


EpiTect Hi-C workflow – day 2

Day 2 of the EpiTect Hi-C workflow.


The EpiTect Hi-C Kit generates high-quality Hi-C NGS libraries, ensuring that first-rate data is generated from costly downstream deep sequencing. Sequencing results from more than 40 EpiTect Hi-C libraries have been analyzed to evaluate the performance of the kit. The most important QC metrics are shown in the following figures: Percentage of Hi-C events, Percentage of long-range cis interactions, Cis/Trans ratio, No strand orientation bias with the EpiTect Hi-C Kit and Percentage of paired reads deriving from a single restriction fragment. The data show that the EpiTect Hi-C Kit generates NGS libraries that, on average, far exceed criteria normally considered sufficient for a successful Hi-C experiment.


Hi-C is a proximity ligation assay that captures chromatin interactions on a genome-wide scale. The EpiTect Hi-C Kit is a specialized DNA preparation resulting in an Illumina-compatible NGS library (see figures EpiTect Hi-C workflow – day 1 and EpiTect Hi-C workflow – day 2). Briefly described, the assay starts with the purification of nuclei in which chromatin conformation has been frozen by chemical cross-linking of DNA binding proteins and DNA. The DNA is then completely digested with a 4 bp restriction enzyme. Open DNA ends are labeled with biotin and subsequently ligated. Paired-end sequencing of the Hi-C ligation products identifies very large numbers of chimeric sequences that derive from DNA strands that were closely associated in space. The probability that two sequences are ligated together is a function of their average distance in space. Quantification of ligation junctions allows for the determination of DNA contact frequencies from which high-resolution mapping of chromatin folding can be achieved.


The EpiTect Hi-C workflow (see figures EpiTect Hi-C workflow – day 1 and EpiTect Hi-C workflow – day 2) represents a marked improvement over published protocols. A week-long and complicated procedure has been converted into a simple and robust protocol that requires just 1.5 days. Furthermore, the sample input requirement has been reduced by one order of magnitude, allowing creation of Hi-C NGS libraries from just 250,000 cells. The protocol has been developed for work with cross-linked cells from mammalian cell cultures.


The EpiTect Hi-C procedure is a version of the in situ (i.e., in nucleus) Hi-C method in which nuclei are gently purified and permeabilized to maintain the spatial organization of the genome during the initial digestion and ligation steps. This process is vital in order to minimize background noise from uninformative ligation events that do not reflect genome organization. This is because intact nuclei constrain the movement and random collisions of cross-linked complexes, such that ligation events predominantly occur between topologically associated DNA fragments.


Constructing Hi-C NGS libraries

The EpiTect Hi-C Kit workflow consists of 2 parts and each can be completed in one day. The steps of the protocol are summarized in the tables below and visualized in figures EpiTect Hi-C workflow – day 1 and EpiTect Hi-C workflow – day 2. The included Illumina adapters have sequence bar codes that enable multiplex sequencing of up to 6 samples.


Hi-C procedure part 1 (day 1):

Step: Duration

Lysis of cross-linked cells: 15 min

Chromatin opening: 10 min

Chromatin digestion: 2.5 h

Hi-C End Labeling: 30 min

Hi-C Ligation: 2 h

De-crosslinking: 2 h

DNA purification: 10 min


Hi-C procedure part 2 (day 2):

Step: Duration

Sonication: 15 min

DNA purification: 15 min

Streptavidin pulldown: 30 min

DNA end-repair/A-tailing: 30 min

Illumina adapter ligation: 1 h

NGS library amplification: 30 min

DNA purification: 15 min


To view the full protocol, see our detailed EpiTect Hi-C Handbook.


Data analysis

Hi-C data analysis is offered at our GeneGlobe Data Analysis Center. Hi-C sequencing results can be analyzed using the EpiTect Hi-C Data Analysis Portal, which uses open-source tools to provide a QC sequencing report, Hi-C contact matrices and visualization of chromatin contact maps. For more information see our EpiTect Hi-C Data Analysis Portal User Guide.


Chromatin conformation

The three-dimensional organization of chromatin is under intense investigation because it has a profound influence on genome function. By enabling the capture of long-range DNA contacts on a genome-wide scale, Hi-C has quickly become a very important tool for the analysis of nuclear organization. Analysis of Hi-C data has revealed the amazing complexity of genome architecture, with multiple layers of spatial organization that partition the genome into chromosome territories, chromosomal sub-compartments, topologically associated domains (TADs) and DNA loops at increasing resolution (see figure Levels of chromatin organization). In addition, genome organization is dynamic and changes during development. In no two cell types do chromosomes fold alike.


Hi-C technology is refining our understanding of the underlying mechanisms of gene regulation by demonstrating the crucial role played by chromatin folding. For example, Hi-C data have provided overwhelming, concrete experimental evidence for the long-held belief that enhancers and cognate promoters are joined together in space by DNA loops, explaining how regulatory elements can exert control of gene expression over great distances of genome sequence. In addition, Hi-C data have revealed that chromatin folding helps establish separate nuclear compartments with distinct regulatory environments by grouping together, in space, DNA domains that are distal in sequence, but which share the same set of epigenetic marks. Importantly Hi-C data is also revealing how mutations that cause subtle changes in chromatin structure can alter gene expression dramatically and cause disease.


Genome assembly – haplotype phasing

Massive international efforts to sequence the genomes of all representative forms of eukaryotic life are under way. While genome sequencing currently benefits from advances in long-read DNA sequencing technologies, the assembly of sequence scaffolds is nevertheless limited by large stretches of repetitive sequence that extend the range of sequencing. Hi-C technology can be used to join pieces of the puzzle. Individual chromosomes are physically separated into discrete territories and, therefore, DNA interactions captured by Hi-C primarily take place between DNA from the same chromosome (in cis) with little interaction between chromosomes (in trans). Additionally, a significant portion of these cis interactions is long range, occurring between loci separated by millions of bases of DNA. These properties of chromatin interactions can be leveraged in de novo genome assembly. Analysis of Hi-C data help order, orient and join sequence scaffolds into near full-length chromosomes, without the need of a reference genome. Similarly, Hi-C interaction maps are used to detect chromosomal rearrangements and to create diploid genomes by assigning genetic variants to paternal and maternal sister chromosomes (see figure Downstream applications of Hi-C sequencing data).

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