Question: How should I proceed with my Fixed RNA Profiling samples if I experience a clog or wetting failure?
Answer: If a clog or wetting failure occurs, these samples can be re-run in a new GEM reaction.
Option A) If additional reagents are available to immediately rerun the GEM reaction, we recommend refiltering the samples with 30 µm filters, prior to rerunning. In rare cases, there may not be sufficient volume to filter (< 500 µl). In this situation, we would recommend diluting the sample to at least 500 µl before filtering, and then spinning down the sample to concentrate as described in the User Guide as needed.
Option B) If additional reagents are not available to immediately rerun the GEM reaction, we recommend storing the samples at -20°C or -80°C using the guidance provided in the User Guide, which is also noted here.
- Add 0.1 volume Enhancer to sample in Post-Hyb Resuspension Buffer. For example, add 50 μl Enhancer to 500 μl of sample in Post-Hyb Resuspension Buffer.
- Add 50% glycerol for a final concentration of 10%. For example, add 137.5 μl 50% glycerol to 550 μl sample in Post-Hyb Resuspension Buffer and Enhancer.
- Store at −20°C or −80°C for up to 1 month. For best results, storage at −80°C is strongly recommended.
Using Stored Samples
When ready to use samples stored at −20°C or −80°C from this step, thaw at room temperature until no ice remains and then continue from step 2.1q of 2.1 Post-Hybridization Pool & Wash on page 54 step to wash the sample once before proceeding to the step 2.2. Samples may undergo a color change during storage (e.g., black, light gray, or green), however this will not impact assay performance.
We strongly recommend rerunning the sample if a clog occurs during GEM generation. In some cases, minor clogs may result in recovery of >90 μl but <100 μl GEMs. Though the cell recovery efficiency might be slightly reduced, it may be possible to continue with the protocol and sequencing to recover information from the rest of the sample.
As part of our warranty policy, clog and wetting failures that have been properly documented are reimbursed with replacement reagents and chips. Please send (1) the lot numbers of the Gel Beads and chips that were used when the failure occurred, (2) a picture of the failed emulsions in the pipette tips/strip tubes and the chip (see this article: How should I take photographs to document clogs and wetting failures?), and (3) a recent purchase order and a contact phone number to firstname.lastname@example.org.
Products: Fixed RNA Profiling