Question: Can I use formalin fixed tissue as input for the Gene Expression Flex assay?
Answer: Based on limited testing, we found that cells and tissues fixed in formalin are compatible with the Gene Expression Flex workflow. Depending on the temperature and length of formalin fixation, variable results in the 10x Genomics assay may be observed. For optimal assay performance, we would recommend to follow our fixation Demonstrated Protocols (Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling or Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling), as these protocols have been robustly tested across many tissue types upstream of the Gene Expression Flex assay.
If precious samples have already been fixed using 10% neutral buffered formalin (10% NBF), we would recommend that the fixative be replaced with Nuclease-free Water when making the 10x Genomics Fixation Buffer with the Conc. Fix & Perm Buffer (PN-2000517). The Fixation Buffer contains a mild detergent which may help permeabilize the sample in preparation for downstream Probe Hybridization. A one hour room temperature incubation should be sufficient for permeabilization of formalin fixed samples.
Some samples fixed in formalin may not require an additional permeabilization step. Unfortunately, we do not currently have any guidelines on which tissue types may or may not require permeabilization. If you elect to skip the abovementioned incubation, ensure to spin the formalin fixed sample down and resuspend using 10x Genomics Quenching Buffer before tissue dissociation (as described in CG000553) or before proceeding with the Chromium Fixed RNA Profiling protocols (as described in CG000478).
Please note: From limited testing, we found that samples fixed with 10% NBF for longer periods of time (~24 hours) at 4C are more likely to generate higher-quality data as compared to samples fixed at room temperature for the same amount of time.
Products: Fixed RNA Profiling, Single Cell Gene Expression Flex