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HiChIP and Hi-C Protocol Optimized for Primary Murine T Cells
Tomas Zelenka1,2, Charalampos Spilianakis1,2
1Department of Biology, University of Crete, GR70013 Heraklion, Crete, Greece.
Methods and Protocols
|July 21, 2021
Summary
This study optimizes HiChIP and Hi-C protocols for T cells, significantly reducing unusable data and improving the detection of long-range chromatin interactions for more reliable 3D genome studies.
Area of Science:
- Genomics
- Molecular Biology
- Epigenetics
Background:
- Three-dimensional genome organization is crucial for cellular function.
- Hi-C and HiChIP are key methods for studying chromatin interactions, but face challenges like dangling ends and protocol variability.
- Chromatin compactness in cell types like T cells complicates genomic analyses.
Purpose of the Study:
- To systematically optimize the HiChIP protocol for primary T cells.
- To improve the efficiency and reproducibility of HiChIP and Hi-C experiments.
- To provide a detailed, optimized protocol for researchers.
Main Methods:
- Systematic optimization of major HiChIP protocol steps in T cells.
- Application of optimized steps to Hi-C experiments.
- Validation using multiple mouse genotypes and biological replicates.
Main Results:
- Reduced dangling ends to near zero, increasing usable reads.
- Significantly increased the proportion of long-range interaction pairs.
- Demonstrated high reproducibility of the optimized protocol.
Conclusions:
- The optimized protocol enhances the reliability and efficiency of HiChIP and Hi-C in T cells.
- This work addresses key limitations in 3D genome analysis, particularly in challenging cell types.
- A detailed, optimized protocol for both HiChIP and Hi-C is provided.
