Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing

Area of Science:

• Biotechnology
• Genomics
• Microfluidics

Background:

• Single-cell RNA sequencing (scRNA-seq) is crucial for understanding cellular heterogeneity.
• Droplet microfluidics enables automated single-cell analysis but faces challenges with barcoding errors and low throughput.
• Existing methods struggle with multiple-beads-in-a-droplet issues and inefficient bead concentrations.

Purpose of the Study:

• To develop a droplet-based microfluidic platform for high-throughput, accurate single-cell expression profiling.
• To overcome barcoding errors and throughput limitations in current scRNA-seq methods.
• To enable precise analysis of cellular heterogeneity.

Main Methods:

• Developed a novel droplet microfluidic platform utilizing deterministic bead encapsulation.
• Employed inertial focusing in a spiral channel to order oligonucleotide barcode-bearing beads.
• Simultaneously encapsulated ordered beads and single cells into droplets.

Main Results:

• Achieved deterministic single-bead-in-a-droplet encapsulation with minimal multiple-beads-in-a-droplet events, even at high bead concentrations (1000 μl⁻¹).
• Significantly reduced barcoding errors, enabling accurate high-throughput barcoding for scRNA-seq.
• Demonstrated that multiple-beads-in-a-droplet events in standard devices can lead to underestimation of transcripts and overestimation of cell numbers.

Conclusions:

• The developed platform significantly improves throughput and accuracy in single-cell RNA sequencing.
• Deterministic bead encapsulation effectively minimizes barcoding errors, enhancing data reliability.
• This technology expands the utility and practicality of droplet-based single-cell analysis.