BACKGROUND Transcriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS). METHODS To address this problem, we systematically analyzed the critical steps, and found that ethanol fixation together with optimized downstream procedures provided a pipeline that yielded high quality total RNA in amounts to readily support the RNA-seq procedure, while still preserving good discrimination between the individual hLESC immunophenotypes. RESULTS The average RNA integrity number (RIN) was 7.7 ± 0.4, and the average yield was 4.6 ± 1.7 pg of RNA per cell. The sequencing analysis of the isolated RNA produced high quality data with more than 70% of read pairs mapping uniformly to the reference genome and 80% of bases with a Phred score of at least 30. CONCLUSION In this study, we developed a reliable FACS-based procedure using ethanol as a fixative that would support accurate isolation of limbal epithelial progenitor subpopulations along with RNA yield and quality sufficient to enable deep transcriptomic profiling.

中文翻译：

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在荧光激活细胞分选 (FACS) 后保持离体培养的角膜缘上皮干细胞转录组分析的 RNA 完整性。

背景 离体培养的人角膜缘上皮干细胞 (hLESC) 的转录组分析将促进更好地了解角膜生理学和角膜缘干细胞缺乏症 (LSCD) 的新治疗范例。然而，目前此类分析研究受到阻碍，因为难以从基于膜和细胞内抗原共表达通过荧光激活细胞分选（FACS ）。方法 为了解决这个问题，我们系统地分析了关键步骤，发现乙醇固定和优化的下游程序提供了一条产生高质量总 RNA 的管道，其数量足以支持 RNA-seq 程序，同时仍然保持个体 hLESC 免疫表型之间的良好区分。结果平均 RNA 完整性数 (RIN) 为 7.7 ± 0.4，平均产量为每个细胞 4.6 ± 1.7 pg RNA。分离的 RNA 的测序分析产生了高质量的数据，超过 70% 的读取对均匀地映射到参考基因组，80% 的碱基的 Phred 评分至少为 30。 结论 在本研究中，我们开发了一种可靠的 FACS-基于使用乙醇作为固定剂的程序，该程序将支持准确分离角膜缘上皮祖细胞亚群以及足以实现深度转录组分析的 RNA 产量和质量。