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The effect of methanol fixation on single-cell RNA sequencing data
Xinlei Wang1, Lei Yu1, Angela Ruohao Wu2,3,4
1Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China.
BMC Genomics
|June 6, 2021
Summary
Methanol fixation for single-cell RNA sequencing (scRNA-seq) preserves biological signals. Fixation biases are subtle and depend on library construction, with Drop-seq showing fewer effects.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Single-cell RNA sequencing (scRNA-seq) advances understanding of tissue heterogeneity.
- Sample fixation is increasingly needed for scRNA-seq in transport or synchronization.
- Methanol fixation is a common, gentle method, but potential biases are a concern.
Purpose of the Study:
- To evaluate the impact of methanol fixation on scRNA-seq data.
- To assess RNA integrity and biological signal preservation after fixation.
- To understand how fixation biases vary with library preparation methods.
Main Methods:
- Adapted methanol fixation protocol for scRNA-seq.
- Performed scRNA-seq on both live and methanol-fixed cells.
- Analyzed fixation effects on transcripts based on length and GC content.
Main Results:
- Methanol fixation faithfully preserves biological signals.
- Fixation-induced discrepancies are subtle and influenced by library construction.
- Transcript features impact fixation sensitivity; Drop-seq shows reduced effects.
Conclusions:
- Methanol fixation has minimal impact on overall biological signals in scRNA-seq.
- Fixation effects are dependent on sequencing library preparation.
- Understanding these effects is crucial for designing scRNA-seq experiments involving fixation.
