The effect of methanol fixation on single-cell RNA sequencing data

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...

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Bibliographic Details
Published in:BMC genomics 2021-06, Vol.22 (1), p.1-420, Article 420
Main Authors: Wang, Xinlei, Yu, Lei, Wu, Angela Ruohao
Format: Article
Language:English
Subjects:
Biological specimens
Chemical properties
Composition
Construction methods
Datasets
Drop-seq
Fixation
Gene sequencing
Genes
Genomics
Heterogeneity
Methanol
Methanol fixation
Principal components analysis
Protection and preservation
Proteins
Ribonucleic acid
RNA
RNA sequencing
Single Cell RNA-seq
Smarts-seq2
Tissues
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Summary: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.
ISSN:1471-2164
1471-2164
DOI:10.1186/s12864-021-07744-6