Isolation of planarian viable cells using fluorescence-activated cell sorting for advancing single-cell transcriptome...

Area of Science:

• Cell Biology
• Genomics
• Molecular Biology

Background:

• Viable single-cell preparation is crucial for accurate single-cell RNA sequencing (scRNA-seq).
• Ambient RNA from non-viable cells can compromise scRNA-seq data integrity.
• Previous methods using UV lasers for cell staining can cause cellular damage.

Purpose of the Study:

• To develop an improved method for isolating viable single cells from planarian bodies for scRNA-seq.
• To optimize fluorescence-activated cell sorting (FACS) for planarian cell isolation.
• To achieve high-quality scRNA-seq data from diverse cell types.

Main Methods:

• Developed a FACS-based method using a violet laser to minimize cellular damage during Hoechst 33342 staining.
• Optimized cell staining conditions and FACS compensation parameters.
• Identified a non-staining FACS gating strategy to exclude low-quality cells and debris.

Main Results:

• Initial attempts with violet laser staining yielded comparable FACS profiles to UV laser methods.
• High-quality scRNA-seq data were obtained for adult pluripotent stem cells (aPSCs).
• A non-staining FACS strategy significantly improved scRNA-seq data quality for all cell types (>80% barcoding mRNA efficiency).

Conclusions:

• The non-staining FACS strategy effectively isolates viable single cells from planarians.
• This method enhances scRNA-seq data quality across diverse cell populations.
• The non-staining FACS approach shows potential for viable cell isolation in various organisms for scRNA-seq studies.