Generating a Three-Dimensional Genome from Xenopus with Hi-C

Area of Science:

• Genomics
• Molecular Biology
• Developmental Biology

Background:

• Three-dimensional (3-D) genome architecture influences gene regulation.
• Hi-C is a sequencing-based method to capture genome-wide interactions.
• Analyzing 3-D genome structure in Xenopus embryos is crucial for understanding development.

Purpose of the Study:

• To describe a streamlined, high-throughput, and cost-effective in situ Hi-C protocol for Xenopus embryos.
• To enable the generation of high-quality Hi-C libraries from small cell numbers.
• To facilitate genome assembly and haplotype phasing in Xenopus.

Main Methods:

• Formaldehyde fixation of Xenopus embryos.
• In situ nuclear lysis, chromatin digestion, biotinylated nucleotide fill-in, and proximity ligation.
• Isolation and sonication of ligated DNA, followed by streptavidin bead capture of biotinylated junctions.
• Ligation-mediated PCR amplification, paired-end sequencing, and bioinformatic analysis.

Main Results:

• A streamlined in situ Hi-C protocol optimized for Xenopus embryos.
• High-quality Hi-C libraries generated from as few as ~10,000 cells in 2 days.
• Demonstrated utility of the method for genome assembly and haplotype phasing in Xenopus.

Conclusions:

• The optimized in situ Hi-C protocol provides a powerful tool for studying 3-D genome organization in Xenopus.
• This method is suitable for high-throughput, low-cost analysis, even with limited biological material.
• The protocol advances research in Xenopus genomics and developmental biology.