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
This protocol has been optimized using Jurkat cells and fresh mouse embryonic neuronal tissue.
Initial experiments comparing gene expression from fresh mouse embryonic neuronal cells and mouse embryonic neuronal cells that were fixed and rehydrated (either the same day or stored in methanol for 6 days at 4°C, −20°C or −80°C) show very comparable data and library complexity, with Pearson correlations ranging from 0.92-0.96. 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.
Products: Single Cell 3'