Question: Can I isolate nuclei from frozen tissue for gene expression profiling?
Answer: Unfortunately, we do not have direct experience isolating nuclei from frozen tissue for Single Cell gene expression profiling.
We do have a demonstrated protocol for the isolation of nuclei from fresh neural tissue and single cell suspensions: https://support.10xgenomics.com/single-cell-gene-expression/index/doc/demonstrated-protocol-isolation-of-nuclei-for-single-cell-rna-sequencing
This protocol describes the best practices and general protocols for cell lysis, washing, debris removal, counting, and concentrating nuclei in preparation for use in the 10x Single Cell protocol.
The isolation of nuclei from frozen tissue may require additional optimization during dissociation and/or cell handling not covered in this protocol.
If possible, we would suggest isolating single nuclei before freezing. We recommend freezing the nuclei suspension in an appropriate buffer for our 10x assay. This online protocol may be useful as a guide: http://collaslab.org/wp-content/uploads/2011/07/Isolation_somatic_cell_nuclei.pdf
You will need to freeze down at least 1 million nuclei total to recover sufficient numbers post thaw since you'll lose some during the wash and centrifugation steps. Note, this is not a 10x-supported protocol and may require some optimization.
Note: Depending on the cell type, sample preparation and cryopreservation may need to be adjusted to preserve cell viability.
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