Collection of cells for single-cell RNA sequencing using high-resolution fluorescence microscopy

Area of Science:

• Single-cell transcriptomics
• Cellular imaging and sorting
• Molecular biology techniques

Background:

• Fluorescence-activated cell sorting (FACS) is widely used for single-cell RNA sequencing (SORT-Seq).
• FACS has limitations, including inability to measure subcellular fluorescence distribution and suitability only for larger cell samples (>1,000 cells).
• The VYCAP puncher system offers a potential solution to these limitations.

Purpose of the Study:

• To describe a novel workflow for capturing, imaging, and collecting fluorescent human retina pigment epithelium cells for SORT-Seq.
• To demonstrate the utility of the VYCAP puncher system for single-cell transcriptomic applications.
• To present a method applicable to various cell types with diameters of approximately 5-50 μm.

Main Methods:

• Utilized the VYCAP puncher system for cell capture and imaging.
• Integrated imaging and collection of fluorescent cells for subsequent single-cell RNA sequencing.
• Developed a workflow compatible with human retina pigment epithelium cells and other cell types within a specific size range.

Main Results:

• Successfully demonstrated a workflow for collecting fluorescent cells using the VYCAP puncher system.
• The described method overcomes FACS limitations regarding sample size and subcellular fluorescence measurement.
• The protocol is adaptable for various cell types with diameters of 5-50 μm.

Conclusions:

• The VYCAP puncher system provides a viable alternative to FACS for preparing samples for SORT-Seq, especially for small cell populations.
• This workflow enhances the capabilities for single-cell transcriptomic studies by enabling imaging and collection of specific fluorescent cells.
• The protocol offers a versatile tool for researchers studying cellular heterogeneity and gene expression at the single-cell level.