Nuclei Isolation from Ocular Tissues of the Embryonic Chicken for Single-Nucleus Profiling

Area of Science:

• Developmental Biology
• Genomics
• Molecular Biology

Background:

• High-quality single-nucleus data requires gentle and efficient nuclei isolation from tissues.
• Optimization is crucial across tissue types to maintain nuclear architecture and nucleic acid integrity.
• Existing protocols may not be ideal for specific tissues like embryonic chicken eyes.

Purpose of the Study:

• To present an optimized workflow for single-nucleus suspension from embryonic chicken ocular tissues.
• To ensure compatibility with downstream single-nucleus RNA and ATAC sequencing.
• To provide a cost-effective and adaptable protocol for researchers.

Main Methods:

• Developed a rapid protocol for isolating aggregate-free nuclei from embryonic chicken eyes.
• Incorporated cryopreservation and fixation stopping points for workflow flexibility.
• Included quality control (QC) checkpoints and demonstrated with commercial kits.
• Validated the use of in silico genotyping for deriving biological replicates.

Main Results:

• Achieved rapid isolation of high-yield, aggregate-free nuclei with preserved nucleic acid integrity.
• Demonstrated compatibility of the nuclei suspension with single-nucleus RNA sequencing (snRNA-seq) and single-nucleus ATAC sequencing (snATAC-seq).
• Showcased the ability to computationally derive biological replicates, reducing costs and enabling sex as a variable.
• Successfully adapted existing in silico genotyping methods for this application.

Conclusions:

• The optimized protocol provides a cost-effective, simple, and effective method for single-nucleus profiling of embryonic chicken eye tissues.
• The workflow is adaptable and can likely be modified for similar tissue types.
• This approach significantly reduces the cost of biological replication in genomic studies.