Q
How to deal with scratches on the chip during experiments?
A
RNA molecules could not be captured anymore in scratched areas. Tissue mounting should avoid scratched areas on the chip. If the area is too large, we suggest switching to a new chip.
Q
What should be paid attention to in reagent mix preparation?
A
Since permeabilization process is sensitive to the pH, please use freshly prepared 0.01N HCl (pH = 2). Do not vortex the Permeabilization Enzyme Mix. Pipette-mix before use. We strongly recommend users to purchase and use the HCl from Sigma Aldrich (2104-50mL).
Q
How should I arrange my experiments if the cryostat and slide dryer are not in the same room?
A
We strongly recommend performing drying immediately after sectioning. Please move your slide dryer to the same room and place it right next to the cryostat. Stereo-seq adapter and thermocycler/thermomixer could be used in replacement of a slide dryer. We strongly recommend users to process tissue sections and perform fixation in the same lab area and only transfer the chips to another location at the end of methanol fixation procedure (while the chips are submerged in methanol).
Q
What should I do if I cannot proceed with imaging immediately after fixing?
A
There is high risk of RNA degradation so please arrange the experiment time accordingly and strictly follow the standard experiment procedure.
Q
Is Stereo-seq Transcriptomics Set compatible with other staining methods besides ssDNA staining?
A
Currently, our set is only compatible with ssDNA fluorescent staining.
Q
What should I do if I cannot proceed with permeabilization immediately after imaging?
A
For manual version A2, the glycerol sealed tissue-containing chip can be stored for up to 4 hr after imaging. If the coverslip has already been removed, then proceed to reverse transcription steps immediately to avoid RNA degradation.
Q
What is the reason for the gradual dimming of fluorescence when adding focus points during imaging?
A
Long-term exposure of excitation light will cause partial fluorescence quenching.
Q
Does incomplete tissue removal affect the results?
A
A small amount of tissue on the chip has little to no effect on the Stereo-seq Transcriptomics Set. However, it can affect Stereo-seq Permeabilization Set. Incomplete tissue removal contributes to strong background during imaging, which affects the judgment of the optimal permeabilization time.
Q
Can the same tissue block be sectioned several times throughout multiple days?
A
The tissue block can be sectioned multiple times when stored at -80℃, but must make sure the RIN of the RNA from the tissue is greater than 7.
Q
How to limit contamination between cutting different samples?
A
It is recommended to replace the blades when cutting different samples, and to clean the sectioning area before the next sample. It is best to clean the sectioning area and brushes with ethanol before and after sectioning to avoid cross-contamination of samples.
