Question: Why was the -20°C optional sample storage recommendation removed from the Fixed RNA Profiling assay?
Answer: We have removed the optional -20°C sample storage conditions from the Chromium Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) assay from the optional post-fixation and post-Hybridization sample storage steps. We have observed that the -20°C sample storage can be more difficult to maintain, due to these freezers being more prone to temperature fluctuations than -80°C freezers. In cases where the temperature is not well-maintained, we have occasionally observed the formation of a gel in the samples during post-storage processing. This is not sample type specific. Although we have determined steps to reverse this when it occurs, we have removed the optional -20°C sample storage recommendations from the Demonstrated Protocols and User Guides to minimize the risk of this occurring and improve your experience when running the assay. For optimal assay performance, fixed samples should be stored at -80°C.
If gel is observed after the first centrifugation step in post-storage processing, perform the following steps to remove it:
- Remove as much of the liquid supernatant as possible but do not remove gel.
- Resuspend sample with gel in 500 µl 0.5X PBS (No BSA, No RNase Inhibitor)
- Heat at 42°C 10 min
- Centrifuge 5 min at 850g in room temperature centrifuge.
- Remove supernatant without touching the pellet.
- Repeat steps 2-5 if gel is still present.
- Resuspend pellet in the appropriate post-storage buffer and proceed with workflow.
The data from these samples seems not to be negatively impacted when the steps above are taken to remove the gel, in our internal limited testing.
Products: Fixed RNA Profiling, Single Cell Gene Expression Flex