Question: Can cryopreserved cells be used for gene expression profiling?
Answer: We have successfully generated Single Cell libraries from cryopreserved human and mouse cell lines and PBMCs. We expect some decrease in viability and cell recovery after thawing. This will primarily depend on the cell integrity.
The amount of cells that are frozen should be higher than the targeted cell recovery rate to account for cell loss when you thaw and prepare cells.
For cell lines, we would recommend consulting our Demonstrated Protocol on Fresh Frozen Human-Mouse Cell Line Mixtures for Single Cell RNA Sequencing (https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-fresh-frozen-human-mouse-cell-line-mixtures-for-single-cell-rna-sequencing)
For primary cells, we would recommend consulting our Demonstrated Protocol on Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing (https://support.10xgenomics.com/single-cell/sample-prep/doc/demonstrated-protocol-fresh-frozen-human-peripheral-blood-mononuclear-cells-for-single-cell-rna-sequencing).
When reviving frozen cells for sample preparation, we recommend following these best practices for sample handling:
- Remove cell debris/dead cells with appropriate filters and/or other methods (e.g. FACS, Miltenyi Dead Cell Removal)
- Remove freezing media through washing steps to avoid carry over of contaminants that may inhibit downstream reactions
- Minimize time between cell thawing and chip loading to avoid premature cell lysis
- Resuspend cells in PBS containing 0.04% BSA
- Re-quantify single cells and visually inspect cells prior to loading to confirm viability and cell concentration and to avoid underloading/overloading and reduce the risk of clogs
Note: Depending on the cell type, sample preparation may need to be adjusted to preserve cell viability.
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