RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding.

Area of Science:

• Biotechnology
• Genomics
• Molecular Biology

Background:

• Single-cell RNA sequencing (scRNA-seq) is crucial for understanding cellular heterogeneity.
• Existing scRNA-seq methods often rely on microfluidics or droplet-based systems, which can be costly and complex.
• There is a need for accessible, instrument-free sample preparation techniques for scRNA-seq.

Purpose of the Study:

• To introduce and validate RevGel-seq, a novel reversible-hydrogel technology for scRNA-seq sample preparation.
• To demonstrate the efficacy of RevGel-seq for various cell types, including fresh cells, pancreatic islet cells, and cardiomyocytes.
• To establish RevGel-seq as an efficient and flexible alternative to existing scRNA-seq preparation methods.

Main Methods:

• RevGel-seq utilizes a reversible hydrogel to immobilize complexes of single cells and barcoded beads.
• Cell lysis is induced by detergent diffusion, followed by RNA capture on barcoded beads.
• The technology is validated using peripheral blood mononuclear cells (PBMCs), pancreatic islet cells, and cardiomyocytes.

Main Results:

• RevGel-seq achieves comparable results to microfluidic-based scRNA-seq technologies for PBMC analysis.
• The hydrogel technology effectively accommodates large cells, such as cardiomyocytes.
• A flexible stopping point was established at the cell lysis step, preserving gene expression profiles for up to two hours.

Conclusions:

• RevGel-seq provides an accessible, instrument-free, and efficient method for scRNA-seq sample preparation.
• The technology offers flexibility in experimental design and sample processing timing.
• RevGel-seq demonstrates broad cell type coverage and is suitable for various applications, including clinical research.