Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China.
Department of Chemical and Biological Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China.
BMC Genomics. 2021 Jun 5;22(1):420. doi: 10.1186/s12864-021-07744-6.
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.
单细胞 RNA 测序（scRNA-seq）技术在理解健康和疾病组织异质性方面取得了显著进展。最近，在许多情况下都需要对 scRNA-seq 样本进行固定，例如当样本需要长途运输时，或者当实验需要时间同步时。甲醇固定是一种简单温和的方法，已在 scRNA-seq 中常规应用。然而，人们仍然担心固定可能会导致偏差，从而改变 RNA-seq 的结果。
我们对现有的甲醇固定方案进行了改编，并对活细胞和甲醇固定细胞进行了 scRNA-seq 分析。结果分析表明，甲醇固定可以忠实地保留生物相关信号，而固定引起的差异是微妙的，与文库构建方法有关。通过基于转录本长度和 GC 含量对其进行分组，我们发现不同特征的转录本在全长测序数据中受固定的影响程度不同，而在 Drop-seq 结果中，这种影响得到缓解。
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。
