Question: How can I improve my cell/nuclei recovery using the Fixed RNA Profiling Assay?
Answer: The following strategies can be implement the to reduce cell/nuclei loss throughout the workflow for the Fixed RNA Profiling assay (i.e., Single Cell Gene Expression Flex):
- Use of low binding microcentrifuge tubes
- Use of a swinging bucket rotor centrifuge - cells will pellet at the bottom of the tube in a tighter pellet thus reducing loss during washes
- Increasing the speed/time of centrifugation to improve pelleting
- Use of a transfer pipette to remove supernatant to reduce the risk of aspirating cells
- Avoid pipetting near the cell pellet
- Leaving up to 30μl of supernatant during wash steps so the cell pellet is not disrupted
The expected cell/nuclei loss from all workflow steps (i.e. sample fixation through post-hybridization washes/filtration to chip loading) is ~10-50%, depending on technique/skill, and cell size. Overall, cell loss will be dependent on many factors including user, cell type, and cell number of the sample.
If the cell yield post-tissue dissociation is low when following the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol, the following strategies can be implemented to increase cell yield:
- Increase the tissue dissociation time.
- If tissue is difficult to dissociate with Liberase TL, Liberase TH (1 mg/mL) may also be used as an alternative enzyme.
- If using an Octo Dissociator, after transferring the dissociated cells from the C tube, perform an additional PBS rinse of the C tube and pass the rinse through the 70 µm strainer in step 2e to collect additional cells.
- If manually dissociating tough tissue, use the back of a 1-ml syringe plunger to push any undissociated tissue pieces through the 70 µm strainer.
We recommend performing a pilot experiment to optimize the dissociation of tissue post-fixation.
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex