Question: How does methanol fixation affect transcriptome data from cells?
Answer: A Demonstrated Protocol for methanol fixation prior to Single Cell 3' sequencing can be found here: https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-methanol-fixation-of-cells-for-single-cell-rna-sequencing
The protocol was demonstrated with Jurkat T lymphocytes, mouse embryonic brain cells, and human peripheral blood mononuclear cells (PMBCs).
Initial experiments comparing gene expression from fresh mouse embryonic neuronal cells and mouse embryonic neuronal cells that were fixed and rehydrated show very comparable data and library complexity, with Pearson correlations ranging from 0.92-0.96 (for fresh vs. fixed PBMCs, we found a Pearson correlation of between 0.86 - 0.88). Note that fixing cells will always compromise the transcriptome to a small degree and reduce the library complexity compared to using fresh cells; however, we have found the overall the expression profiles and cell clustering comparable.
It is important to note that data quality from fixed samples may vary by sample type. Our protocol is expected to be compatible with many, but not all, cell or tissue types. Cell and tissue types that are particularly fragile and challenging to dissociate (e.g. organoids, biopsies, primary cancer cells, solid tissue) may require additional optimization to help to preserve the integrity of the transcriptome.
Best practices for performing methanol fixation:
- Treat cells very gently. Methanol fixation is stressful and harsh; rough sample handling on top of that can cause cell lysis
- Cell viability going into methanol fixation is still important (ideally >90%, but greater than 70% is okay).
- You want to work quickly but gently once the cells are out of the fixative. Once they are woken up, transcripts can start leaking out, so ideally you want to move them as quickly as possible out of the fixative, into the wash-resuspension buffer and into the 10x assay before too much leakage occurs.
- Finally, too much wash-resuspension buffer into the RT master mix may inhibit the RT enzyme. We recommend minimizing carryover as much as possible.
Products: Single Cell Gene Expression