Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA Sequencing

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Single-cell and single-nucleus sequencing (sc/sn-seq) are powerful tools in biological research.
• Existing sample barcoding methods face challenges like inhomogeneous expression and limited affinity.

Purpose of the Study:

• To develop a universal sample multiplexing method for sc/sn-seq that overcomes current limitations.
• To introduce Toti-N-Seq, a novel N-glycan-based barcoding technology.

Main Methods:

• Developed Toti-N-Seq using an engineered streptavidin-Fbs1 GYR variant fusion protein to tag N-glycans on cell/nuclear membranes.
• Applied the method to both single-cell sequencing (sc-seq) and single-nucleus sequencing (sn-seq).
• Targeted ubiquitous N-glycans to minimize sample exchange and biased barcoding.

Main Results:

• Achieved high classification accuracy: 0.969 for sc-seq and 0.987 for sn-seq.
• Demonstrated rapid, one-step sample preparation (<3 min) with Toti-N-Seq.
• Successfully detected rare cell populations (~0.5%) in clinical samples while maintaining high fidelity.

Conclusions:

• Toti-N-Seq provides a versatile and efficient platform for universal sample barcoding in sc/sn-seq.
• The technology is broadly applicable across multiple species and eukaryotic cell types.
• Toti-N-Seq minimizes artifacts and batch effects, enhancing the reliability of sequencing data.