High-throughput full-length single-cell mRNA-seq of rare cells

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Single-cell mRNA sequencing (mRNA-seq) offers insights into cancer biology, but low throughput limits its application.
• Current methods struggle with analyzing rare cell populations due to low cell density processing.

Purpose of the Study:

• To develop a high-throughput protocol for rare cell enrichment and single-cell mRNA sequencing.
• To enable differentiation of heterogeneous rare cells by both phenotype and genetic variants.

Main Methods:

• A novel protocol combining a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment.
• Utilized Smart-seq2 chemistry for high-quality, full-length mRNA sequencing.
• Evaluated the protocol using simulated circulating tumor cells (non-small-cell lung cancer lines) spiked into whole blood.

Main Results:

• Achieved high efficiency (>90%) in capturing and releasing simulated rare cells using the magnetic sifter.
• Generated reproducible transcriptome data from enriched rare cells.
• Demonstrated variant analysis from full-length mRNA-seq data to differentiate cells by phenotype and mutation profile in a single run.

Conclusions:

• The developed high-throughput protocol significantly enhances the analysis of rare cell populations.
• This method provides high-quality transcriptomic data and facilitates variant analysis for cell differentiation.
• Enables single-cell characterization of rare cells like circulating tumor cells with improved throughput and detail.