EpiTect Hi-C Kit

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

Features

  • 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
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EpiTect Hi-C Kit (6)

Cat. No. / ID: 59971

Reagents for generating Hi-C NGS libraries for up to 6 samples
€1,975.00
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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.
✓ 24/7 automatic processing of online orders ✓ Knowledgeable and professional Product & Technical Support ✓ Fast and reliable (re)-ordering

Product Details

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.

Performance

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.

See figures

Principle

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.

See figures

Procedure

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.

See figures

Applications

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).

See figures

Supporting data and figures

Resources

Software User Guides (1)
Quick-Start Protocols (1)
Kit Handbooks (1)
EpiTect Hi-C Handbook
PDF (551KB)

FAQ

Does QIAGEN provide data analysis to accompany the EpiTect Hi-C Kit?
Yes. Visit QIAGEN’s online GeneGlobe Data Analysis Center at www.qiagen.com/DataAnalysisCenter to analyze your data with the EpiTect Hi-C Data Analysis Portal.
FAQ ID - 143075
How many reactions are in a kit?
Each EpiTect Hi-C Kit comes with enough reagents for 6 samples. One sample is typically sufficient for the generation of ~300–600 million raw read-pairs.
FAQ ID - 143067
Are there stopping points in the Hi-C workflow?
Yes. Following the Hi-C Digestion, Hi-C End Labeling, Hi-C Ligation, or Chromatin De-crosslinking steps in the Hi-C Part 1 workflow, users can store samples at −15 to −25°C and resume with the procedure at a later date.
FAQ ID - 143071
What kind of analysis does the EpiTect Hi-C Data Analysis Portal perform?
Raw sequencing reads are processed to generate a sequencing report and Hi-C contact matrices. Upon completion of the data analysis, an installation of HiGlass within GeneGlobe can be used to visualize and interact with the generated contact matrices. For further information, refer to the user guide found at the EpiTect Hi-C Data Analysis Portal.
FAQ ID - 143076
Can I substitute the endonuclease used in the Hi-C Digestion step with another endonuclease?
No. The EpiTect Hi-C Digestion Enzyme and Digestion Buffer have been developed to work together to provide optimal results.
FAQ ID - 143070
Can I use cells that have been previously crosslinked and frozen (e.g., for a ChIP or ChIP-seq experiment) as input material for the EpiTect Hi-C protocol?
Yes. However, the cells should have been crosslinked with 1% formaldehyde and not have been frozen for longer than 6 months.
FAQ ID - 143069
With which platform can I perform my sequencing?
EpiTect Hi-C sequencing libraries are compatible with all Illumina sequencing platforms.
FAQ ID - 143072
Can I use my own pipeline to analyze results from my EpiTect Hi-C experiments?
Yes. For your analysis, you will need the following additional information. The endonuclease used in the EpiTect Hi-C kit cuts at GATC sites. The Hi-C junction motifs (or tags) generated after Hi-C ligation consist of a GATCGATC sequence.
FAQ ID - 143078
With which read length should the EpiTect Hi-C NGS libraries be sequenced
Read lengths of 36–150 bp can be used to sequence the EpiTect Hi-C libraries on Illumina sequencers. However, as mappability increases with read length, we suggest the use of 2 x 150 bp sequencing reads for best results.
FAQ ID - 143073
Does the EpiTect Hi-C Data Analysis Portal cost money?
No. EpiTect Hi-C Kit customers may analyze their Hi-C sequencing results with the Data Analysis Portal at no charge.
FAQ ID - 143077
How can I accurately determine the amount of input material for the EpiTect Hi-C Kit?
An automated cell counter or hemocytometer should be used to assist in distributing the correct amount of cells to each sample.
FAQ ID - 143065
What sample source do you support?
In principle the EpiTect Hi-C protocol should work for any type of crosslinked eukaryote cells; but so far, it has only been experimentally validated with cultured human and mouse cells. Bacteria and archaea are not supported.
FAQ ID - 143066
What is the effect of using amounts of input material per sample that fall outside the recommended range?
Using input amounts outside the range of 2.5 x 105 and 2.5 x 106 human or mouse cells (or the equivalent of 1.5–15 µg DNA) per sample risks affecting the quality of the resulting NGS library, such that it is likely to have higher levels of inward-facing (FR) sequence read bias, unligated ends, and read pairs containing contiguous, religated restriction fragments.
FAQ ID - 143064 
What is the required amount of input material per sample for the EpiTect Hi-C Kit?
The EpiTect Hi-C protocol has been optimized for the use of 5 x 105 human or mouse cells (or the equivalent of 3 µg of DNA) per sample. Input amounts between 2.5 x 105 and 2.5 x 106 human or mouse cells (or the equivalent of 1.5–15 µg DNA) per replicate may also be used.
FAQ ID - 143063
How long does it take to complete the EpiTect Hi-C protocol?
From crosslinked sample to purified Illumina NGS library, the EpiTect Hi-C protocol takes 1.5–2 days to complete.
FAQ ID - 143068
How do I know if my Hi-C sample is a good candidate for deep sequencing?
Prior to costly deep sequencing, users are advised to sequence Hi-C NGS libraries at low depth (<1 million reads) for quality control purposes. Low-depth sequencing data can be processed with the EpiTect Hi-C Data Analysis portal at www.qiagen.com/DataAnalysisCenter, and the sequencing report can be used to assess the quality of Hi-C libraries.
FAQ ID - 143074