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.

中文翻译：

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使用荧光激活细胞分选分离涡虫活细胞以推进单细胞转录组分析

准备可行的单细胞对于进行单细胞 RNA 测序 (scRNA-seq) 至关重要，因为来自死亡或受损细胞的环境 RNA 的存在会干扰数据分析。在这里，我们开发了一种使用荧光激活细胞分选（FACS）从成年涡虫体内分离活单细胞的方法。然后将该方法应用于成体多能干细胞 (aPSC) 和分化/分化细胞。最初，我们使用紫激光而不是紫外 (UV) 激光来激发 Hoechst 33342，以减少细胞损伤。在优化细胞染色条件和 FACS 补偿后，我们生成的 FACS 图谱与使用之前使用 UV 激光的方法创建的图谱类似。尽管成功获得了 aPSC 的高质量 RNA 测序数据，但非 aPSC 产生了低质量的 RNA 读数（即，<60% 的细胞拥有条形码 mRNA）。随后，我们确定了一种有效的 FACS 门控条件，无需染色即可排除低质量细胞和组织碎片。这种非染色分离策略不仅缩短了解离后时间，而且还为所有细胞类型（即 >80%）提供了高质量的 scRNA-seq 结果。总而言之，这些发现表明，非染色 FACS 策略可能不仅有利于从涡虫中分离活细胞，而且有利于从其他生物和组织中分离活细胞以进行 scRNA-seq 研究。