Single-Cell Digital Lysates Generated by Phase-Switch Microfluidic Device Reveal Transcriptome Perturbation of Cell...

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Conventional gene expression profiling averages cellular heterogeneity.
• Single-cell transcriptome analysis offers potential but requires determining optimal cell numbers.
• Assessing cellular heterogeneity is crucial for understanding biological systems.

Purpose of the Study:

• Introduce and validate the concept of "digital lysate" for assessing cellular heterogeneity.
• Develop a method to construct a molecular map of transcriptome perturbation during the cell cycle.
• Determine the necessary number of cells for meaningful single-cell study conclusions.

Main Methods:

• Utilized a phase-switch droplet microfluidic platform for random single-cell transcriptome sampling.
• Generated "digital lysates" by permuting and averaging multiple single-cell transcriptomes.
• Employed next-generation sequencing for transcriptome analysis and siRNA assays for validation.

Main Results:

• Digital lysates closely converged with physical lysates (r = 0.93).
• Achieved high sample-to-sample repeatability comparable to conventional methods (r = 0.98).
• Successfully mapped cell cycle dynamics and inferred cell cycle regulatory genes.

Conclusions:

• The "digital lysate" approach effectively assesses cellular heterogeneity.
• This method provides a robust framework for molecular mapping and gene regulatory inference.
• The study establishes an effective strategy for analyzing single-cell transcriptome data.