A SINC-Seq Protocol for the Analysis of Subcellular Gene Expression in Single Cells

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Microfluidic devices enable precise control of single cells and molecules.
• Current methods face limitations in subcellular RNA analysis and contamination.
• Single-cell resolution is crucial for understanding cellular heterogeneity.

Purpose of the Study:

• To introduce a novel microfluidic approach for precise fractionation of cytoplasmic and nuclear RNA from single cells.
• To enable detailed analysis of gene expression and RNA localization within subcellular compartments.
• To provide a comprehensive protocol for SINC-seq compatible with short-read and long-read sequencing.

Main Methods:

• Development of a microfluidic system utilizing a hydrodynamic trap and electric field control.
• Isolation of single cells and selective lysis of the plasma membrane.
• Electrophoretic extraction of cytoplasmic RNA while retaining the nucleus.
• Off-chip library preparation for RNA sequencing (Illumina and Oxford Nanopore Technologies).

Main Results:

• Demonstration of precise fractionation of cytoplasmic and nuclear RNA from individual cells.
• Successful application of electric field control for subcellular manipulation within microfluidics.
• Generation of RNA sequencing libraries for both short-read and long-read platforms.

Conclusions:

• SINC-seq offers a powerful tool for dissecting gene expression and RNA localization at the subcellular level.
• This microfluidic-based approach enhances resolution and minimizes contamination in single-cell RNA analysis.
• The protocol facilitates comprehensive transcriptome analysis from distinct cellular compartments.