A protocol to study three-dimensional genome structure in individual mutant preimplantation mouse embryos

Area of Science:

• Genomics
• Developmental Biology
• Molecular Biology

Background:

• The three-dimensional genome structure influences gene regulation.
• High-throughput chromosome conformation capture (Hi-C) is a key technique for studying genome architecture.
• Genotyping mouse embryos is crucial for genetic studies.

Purpose of the Study:

• To develop and describe a single-blastocyst Hi-C protocol for genotyping mutant mouse embryos.
• To enable sequence-based genotyping of individual embryos using Hi-C data.
• To provide a detailed methodology for researchers in developmental biology and genomics.

Main Methods:

• Single-blastocyst Hi-C protocol development.
• Embryo fixation and nuclei permeabilization.
• Chromatin digestion, biotin-labeled nucleotide incorporation, and re-ligation.
• Purification of chimeric DNA ligations.
• Library generation, sequencing, and genome-wide interaction analysis.

Main Results:

• Successful implementation of single-blastocyst Hi-C for mutant mouse embryo genotyping.
• Detailed protocol steps for chromatin conformation capture and analysis.
• Demonstration of genome-wide interaction analysis from single cells.

Conclusions:

• Single-blastocyst Hi-C is a viable method for sequence-based genotyping of mutant mouse embryos.
• This protocol provides a powerful tool for studying the genetic basis of early embryonic development.
• The methodology facilitates high-resolution analysis of genome 3D organization in individual embryos.