Single-cell RNA sequencing (scRNA-seq) has led to remarkable progress in our understanding of tissue heterogeneity in health and disease. Recently, the need for scRNA-seq sample fixation has emerged in many scenarios, such as when samples need long-term transportation, or when experiments need to be temporally synchronized. Methanol fixation is a simple and gentle method that has been routinely applied in scRNA-seq. Yet, concerns remain that fixation may result in biases which may change the RNA-seq outcome. We adapted an existing methanol fixation protocol and performed scRNA-seq on both live and methanol fixed cells. Analyses of the results show methanol fixation can faithfully preserve biological related signals, while the discrepancy caused by fixation is subtle and relevant to library construction methods. By grouping transcripts based on their lengths and GC content, we find that transcripts with different features are affected by fixation to different degrees in full-length sequencing data, while the effect is alleviated in Drop-seq result. Our deep analysis reveals the effects of methanol fixation on sample RNA integrity and elucidates the potential consequences of using fixation in various scRNA-seq experiment designs.

中文翻译：

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甲醇固定对单细胞RNA测序数据的影响

单细胞RNA测序（scRNA-seq）已使我们对健康和疾病中组织异质性的理解有了显着进步。最近，在许多情况下都出现了对scRNA-seq样品固定的需求，例如，当样品需要长期运输时，或者当实验需要时间同步时。甲醇固定是一种简单而温和的方法，已常规应用于scRNA-seq。然而，仍然存在着担忧，即固定可能导致偏差，从而可能改变RNA-seq的结果。我们适应了现有的甲醇固定方案，并在活细胞和甲醇固定细胞上进行了scRNA-seq。结果分析表明，甲醇固定可以忠实地保留生物学相关信号，而固定引起的差异则微妙且与文库构建方法有关。通过基于转录物的长度和GC含量对转录物进行分组，我们发现全长测序数据中具有不同特征的转录物受固定程度不同的影响，而Drop-seq结果的影响有所减轻。我们的深入分析揭示了甲醇固定对样品RNA完整性的影响，并阐明了在各种scRNA-seq实验设计中使用固定的潜在后果。