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

Preparing viable single cells is critical for conducting single-cell RNA sequencing (scRNA-seq) because the presence of ambient RNA from dead or damaged cells can interfere with data analysis. Here, we developed a method for isolating viable single cells from adult planarian bodies using fluorescence-activated cell sorting (FACS). This method was then applied to both adult pluripotent stem cells (aPSCs) and differentiating/differentiated cells. Initially, we employed a violet instead of ultraviolet (UV) laser to excite Hoechst 33342 to reduce cellular damage. After optimization of cell staining conditions and FACS compensation, we generated FACS profiles similar to those created using a previous method that employed a UV laser. Despite successfully obtaining high-quality RNA sequencing data for aPSCs, non-aPSCs produced low-quality RNA reads (i.e., <60% of cells possessing barcoding mRNAs). Subsequently, we identified an effective FACS gating condition that excluded low-quality cells and tissue debris without staining. This non-staining isolation strategy not only reduced post-dissociation time but also enabled high-quality scRNA-seq results for all cell types (i.e., >80%). Taken together, these findings imply that the non-staining FACS strategy may be beneficial for isolating viable cells not only from planarians but also from other organisms and tissues for scRNA-seq studies.