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FACS-Based Sequencing Approach to Evaluate Cell Type to Genotype Associations Using Cerebral Organoids
Liam Murray1,2,3,4, Meagan N Olson1,2,3,4, Nathaniel Barton1,2,3,4
1Program in Bioinformatics and Integrative Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|June 10, 2023
Summary
This study presents a new method using CRISPR/Cas9 edited cerebral organoids and fluorescence-activated cell sorting (FACS) to link genotypes with specific cell types. This high-throughput approach enables accurate validation of genotype-to-cell type associations.
Area of Science:
- Genomics
- Developmental Biology
- Neuroscience
Background:
- Large-scale transcriptomics sequencing is crucial for identifying genotype-to-cell type associations.
- CRISPR/Cas9 gene editing has advanced the study of cellular function and development.
- Cerebral organoids serve as a model system to study human brain development and disease.
Purpose of the Study:
- To develop and validate a novel fluorescence-activated cell sorting (FACS)-based sequencing method.
- To identify and validate genotype-to-cell type associations using CRISPR/Cas9 edited mosaic cerebral organoids.
- To establish a high-throughput, quantitative approach with internal controls for robust experimental comparisons.
Main Methods:
- Utilizing CRISPR/Cas9 gene editing to create mosaic cerebral organoids with distinct genotypes.
- Employing fluorescence-activated cell sorting (FACS) to isolate specific cell populations based on marker expression.
- Performing high-throughput transcriptomics sequencing on sorted cells to analyze gene expression profiles.
- Implementing internal controls for quantitative comparisons across experiments and antibody markers.
Main Results:
- Successful identification of genotype-to-cell type associations in cerebral organoids.
- Demonstration of the method's high-throughput and quantitative capabilities.
- Validation of the accuracy and reliability of the FACS-based sequencing approach.
- Establishment of a framework for comparing results across different experimental conditions and markers.
Conclusions:
- The described FACS-based sequencing method provides a powerful tool for linking genotypes to cell types.
- This approach significantly advances the study of cellular heterogeneity and genetic contributions in complex tissues.
- The method offers a validated, quantitative, and high-throughput solution for genotype-to-cell type association studies.
