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):
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):
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.
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.
Phase Genomics, Inc, offers a comprehensive suite of computational analytic services for Hi-C data analysis using both open source as well as proprietary software. Services include data QC, Hi-C matrix calculation and visualization, identification of architectural features such as A/B compartments, TADs and loops, structural variation analysis, genome assembly and haplotype phasing, and other custom analyses. A more detailed description of analytic options can be found at phasegenomics.com and a list of publications can be found at Publications & News.