Microdissection and Single-Cell Suspension of Neocortical Layers From Ferret Brain for Single-Cell Assays

Area of Science:

• Neuroscience
• Developmental Biology
• Genetics

Background:

• Brain development involves complex layering of the cerebral cortex.
• Traditional bulk-sequencing methods limit genetic analysis resolution.
• Studying human cortical development requires suitable animal models.

Purpose of the Study:

• To detail a methodology for high-resolution genetic analysis of the developing cerebral cortex.
• To enable the study of specific germinal layers during critical developmental time points.
• To provide a protocol for utilizing advanced single-cell assays on ferret brain tissue.

Main Methods:

• Microdissection of individual germinal layers from living ferret brain slices.
• Enzymatic and mechanical tissue dissociation to create single-cell suspensions.
• Application of single-cell RNA sequencing (scRNA-seq), single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq), and TrackerSeq.

Main Results:

• The protocol yields high-quality single-cell suspensions from embryonic and early postnatal ferret cortex.
• This method allows for increased resolution in genetic analyses compared to bulk sequencing.
• The approach is optimized for ferrets, which better model human cortical development than mice.

Conclusions:

• This microdissection and single-cell analysis protocol enhances the study of complex brain development.
• It provides a powerful tool for investigating genetic regulation in specific cortical layers.
• The methodology facilitates comparative studies using ferrets to model human developmental processes.