Question: How can I improve the yield of my nuclei for hard-to-dissociate tissues such as heart, muscle, and fibrous tissue using the Chromium Nuclei Isolation Kit?
Answer: We recommend not to exceed 2 minutes for the mechanical dissociation with the plastic pestle. If dissociation takes longer than 2 minutes, we recommend the following:
- Mince the frozen tissue on a glass (not plastic) petri dish using pre-chilled razor blades and forceps, keeping it as cold as possible.
- Add the tissue to the tube, add the lysis buffer, and then use 10-15 strokes of the pestle. If this is insufficient, it can be increased up to 25 strokes.
- Optimize the lysis time: Perform a lysis timeline to determine appropriate lysis incubation time for new tissue types. Try 3, 5, and 8 minutes- checking under high magnification (40x or 60x) to determine the quality of nuclei suspension and extent of lysis. It is better to have a slightly under-lysed suspension rather than one that is over-lysed, as the tissue will continue to sit in the lysis buffer for a few more minutes during spin down of the column.
- Check the quality of the nuclei after the final wash to determine whether the lysis time needs to be further adjusted.
- Once the timing is optimized, you can run a test run. If you are running more than one sample, chop all of the tissue pieces first on dry ice, then transfer to the tube with 200µL of lysis buffer on wet ice. Mechanically dissociate each one with the pestle. When the first sample has been dissociated with the supplied pestle in lysis buffer, start the timer. It is recommended to run only 4 samples at a time if it takes longer than 1 minute to dissociate each sample.
- Sample cleanup steps: Additional washes may further reduce small debris and ambient RNA in the sample. If there is still debris present after the final wash, a final filtration step with a 30µm strainer is recommended to remove remaining large debris.
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