A. Use wide-bore pipette tips to avoid damage when handling tissue or nuclei, and pipette slowly.
B. Keep tissue on dry ice until ready to start gentleMACS.
C. Miltenyi gentleMACS Recommendations.
Tissue 1-4 mg - 4C_nuclei_1
Tissue 5-7 mg - cus_4C_nuclei_5
Tissue 7-15 mg - cus_4C_nuclei_10
The "cus" protocols are custom SOP from Miltenyi. It adds 5 or 10 minutes of mixing at 20 RPM after the Miltenyi 5-minute disassociation SOP 4C_nuclei.
* Optimization may be needed depending on tissue type, size, and condition.
D. "To sucrose" or "not to sucrose".
If initial sample size is < 5 mg, it is recommended to wash nuclei without using a sucrose gradient (Step 3). If initial sample size is > 5 mg, it is recommended to purify nuclei using a sucrose gradient (Step 2).
E. Sucrose column recommendations.
After the sucrose cleanup, a tissue less than 3-5 mg may have a very low nuclei yield. It is recommended to test this first on a representative sample to ensure precious samples are not lost.
Hoechst/Syto RNA Select Staining Guidelines
1. Extracellular debris should be minimal after the nuclei have been passed through the sucrose column.
2. Extracellular debris will be present in a much greater quantity if a sucrose column is not used. Debris can interfere with downstream 10x operations by introducing clogs and background ambient RNA.
3. Nuclei should be intact, with minimal blebbing and clumping. Blebbing of nuclei will make them more prone to clumping, which can introduce clogs when loading the 10x chip.
4. The Hoechst and Syto Select staining should be uniform through the nuclei, but the Syto staining usually has more variation.
If working with human tissue, follow all institutional rules and guidelines.