Question: What are the validated stopping points for sample storage in the Fixed RNA Profiling assay?
Answer: Fixed samples can be stored at multiple validated stopping steps within the Fixed RNA Profiling (i.e., Single Cell Gene Expression Flex) workflow.
Sample Preparation:
Fixation of cells or nuclei suspensions: Cells and nuclei suspensions are fixed in a 4% formaldehyde fixative solution and then quenched following the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling (CG000478) Demonstrated Protocol. The Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics PN-1000414) is required for fixation. Fixed cell or nuclei suspensions can be stored short-term at 4°C for up to 1 week or long-term at -80°C for up to 6 months, after resuspending in appropriate reagents.
Fixation and dissociation of tissues: Fresh or flash frozen tissue samples are first fixed and then dissociated following the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling (CG000553) Demonstrated Protocol. Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics PN-1000414) is required for fixation. Fixed tissue pieces and cell suspension derived from fixed dissociated tissues can be stored short-term at 4°C for up to 1 week or long-term at -80°C for up to 6 months, after resuspending in appropriate reagents.
Isolation of Cells from FFPE Tissue Sections: Cells isolated from formaldehyde fixed & paraffin embedded (FFPE) tissue sections can also be used for Chromium Fixed RNA Profiling following the Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling (CG000632) Demonstrated Protocol. Dissociated cells resuspended in appropriate buffer can be stored for short-term at 4°C for up to 1 week or long-term at −80°C for up to 6 months.
Post-Hybridization:
Samples can be optionally stored at two steps within the Fixed RNA Profiling workflow for up to 6 months at -80°C:
Post-Hybridization Pool & Wash Step: After completion of the post-Hyb washes, the samples can be either stored at −80°C for up to 6 months after adding the appropriate reagents, or proceed immediately to the next step. If directly proceeding with the next step, the undiluted washed samples can be placed on ice and then stored later after GEM Incubation.
GEM incubation: The remaining undiluted sample from Post-Hybridization Pool & Wash that has been stored on ice during GEM generation and incubation at −80°C for up to 6 months, after adding the appropriate reagents.
Please note: Samples may undergo a color change during storage (e.g. black, light gray, or green), however this will not impact assay performance.
Products: Fixed RNA Profiling, Single Cell Gene Expression Flex