Nanocoding: Lipid Nanoparticle Barcoding for Multiplexed Single-Cell RNA Sequencing.

Area of Science:

• Biotechnology
• Genomics
• Molecular Biology

Background:

• Single-cell RNA sequencing (scRNA-seq) is powerful but costly and susceptible to batch effects.
• Current sample multiplexing methods using DNA labels have limitations in stability and labeling efficiency, especially in heterogeneous cell populations.
• Efficient multiplexing is crucial for reducing costs and improving data quality in scRNA-seq.

Purpose of the Study:

• To introduce Nanocoding, a novel method for high-density barcode labeling in multiplexed scRNA-seq using lipid nanoparticles (LNPs).
• To demonstrate the efficiency, stability, and broad applicability of Nanocoding across various cell types and sample complexities.
• To showcase Nanocoding's utility in investigating biological questions, such as gene expression changes in aging adipose tissue.

Main Methods:

• Development of Nanocoding utilizing LNPs for cellular uptake and barcode delivery.
• Application of Nanocoding to cultured cell lines, tissue digests (spleen, adipose), and challenging samples with lipid-rich debris.
• Optimization of Nanocoding for rapid labeling (40 minutes) and high barcode loading efficiency (10-100 fold amplification).

Main Results:

• Nanocoding achieved high labeling efficiency (>95%) even in challenging adipose tissue samples from obese rodents.
• The method demonstrated stability after sample mixing and required only commercially available reagents.
• Nanocoding successfully identified cell subtypes in complex tissues and enabled profiling of cells missed by conventional methods.

Conclusions:

• Nanocoding provides a robust and efficient solution for whole-sample multiplexing in scRNA-seq.
• The LNP-based approach overcomes limitations of existing DNA-labeling techniques, offering high barcode density and stability.
• Nanocoding facilitates the study of complex biological systems and cell populations previously difficult to analyze.