TITLE: Nuclei Preparation from Frozen Tissue for 10X Multiome using Dounce Homogenization, Iodixanol Gradient Centrifugation, and FANS (v1.2, Jan 2024)
AUTHORS: ereisner

[DESCRIPTION]
This protocol describes isolation of nuclei from frozen tissue using dounce homogenization, iodixanol gradient centrifugation, and FANS. Nuclei are permeabilized, washed, and counted; single-nucleus suspensions of sufficient concentration and nuclei quality may then be processed using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338, Rev F) protocol from 10X Genomics.

[STEPS]
SECTION: Reagent Preparation
1. Prepare stock Diluent Buffer (1 mL) and 50% iodixanol (6 mL) at room temperature, if needed.

 
 
 
  
 
 

 

SECTION: Reagent Preparation
2.  on ice Prepare all other buffers fresh on ice.

 
 
  
 
  
 
  
 
  
 
  
 
  
 
  
 
  
 
 
SECTION: Nuclei Preparation
3. Pre-chill a large, swing-bucket tabletop centrifuge to 4°C.

SECTION: Nuclei Preparation
4. Retrieve a 1 mL dounce homogenizer with 2 pestles (“Loose” and “Tight”) for each sample. Place on ice and allow to chill.

SECTION: Nuclei Preparation
5. Add 1 mL of NIM-DP-L buffer to each dounce homogenizer.
SECTION: Nuclei Preparation
5.1. **If tissue mass is very small (< 50 mg), instead add 600 µL of NIM-DP-L.
SECTION: Nuclei Preparation
6. Transfer each sample to a dounce homogenizer.
SECTION: Nuclei Preparation
7. Using the loose pestle, gently homogenize the sample until most of the tissue has broken into small pieces (usually 5-10 strokes). 
SECTION: Nuclei Preparation
8. Switch to the tight pestle and homogenize until the solution is uniform with no obvious particles (usually 15-25 strokes). Be gentle and avoid introducing bubbles. 
SECTION: Nuclei Preparation
9. Transfer the full volume of homogenized sample to a 30 μm filter in a 1.5 mL Eppendorf Lobind tube.
SECTION: Nuclei Preparation
10. Rinse each dounce with 1 mL of NIM-DP buffer and transfer the rinse to the filter.
SECTION: Nuclei Preparation
11. Centrifuge for 10 mins at 1000 rcf, 4°C, and 3/3 acceleration/deceleration.
SECTION: Nuclei Preparation
11.1. **If tissue mass is very small (< 50 mg), skip steps 10-11**
SECTION: Nuclei Preparation
12. Discard the supernatant and resuspend the pellet in 1 mL of NIM-DP.
SECTION: Nuclei Preparation
13. Centrifuge for 10 mins at 1000 rcf, 4°C, and 3/3 acceleration/deceleration.
SECTION: Nuclei Preparation
14. Discard the supernatant and resuspend the pelleted nuclei in 2 mL of 20% iodixanol.

SECTION: Nuclei Preparation
16. Centrifuge for 30 mins at 4000 rcf, 4C, and 3/1 acceleration/deceleration.

SECTION: Nuclei Preparation
14.1. **If tissue mass is very small (< 50 mg), instead resuspend in 400 uL of 50% iodixanol (for a final iodixanol concentration of 20%).
SECTION: Nuclei Preparation
15. Slowly pipette the suspension dropwise onto a 500 µL cushion of 25% iodixanol in a 5 mL Eppendorf Lobind tube. **Do not mix this solution once transferred.**
SECTION: Nuclei Preparation
17. Discard the supernatant, leaving a small volume (< 20 µL) to avoid disturbing the pellet.
SECTION: Nuclei Preparation
18. Resuspend the pellet in 500 µL of sort buffer and incubate on ice for 10 mins, protected from light.
SECTION: Nuclei Preparation
19. Sort 120,000-130,000 nuclei into a 1.5 mL Eppendorf Lobind tube containing 90 μL of collection buffer using a Sony SH800 Cell Sorter.
SECTION: Nuclei Preparation
20. Centrifuge the sorted nuclei for 5 mins at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
SECTION: Nuclei Preparation
21. Discard the supernatant.
SECTION: Nuclei Preparation
22. Resuspend the pellet in 100 μL of NPB. Incubate on ice for 1 minute.
SECTION: Nuclei Preparation
23. Add 900 μL of wash buffer.
SECTION: Nuclei Preparation
24. Centrifuge for 5 mins at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
SECTION: Nuclei Preparation
25. Carefully remove the supernatant, leaving 10-15 μL to avoid disturbing the pellet. 
SECTION: Nuclei Preparation
26. Gently resuspend in 12 μL of 1X Nuclei Buffer (prepared from 10X Genomics Multiome protocol).
SECTION: Nuclei Preparation
27. Stain an aliquot of nuclei with 0.4% Trypan Blue. Load 10 μL into one chamber of a hemocytometer.
SECTION: Nuclei Preparation
28. Count nuclei in four quadrants. Average the count and determine the nuclei concentration (nuclei/μL).
SECTION: Nuclei Preparation
29. Capture images from the microscope field at 10X and 20X magnification. Assess nuclei quality.
SECTION: Nuclei Preparation
30. Follow the 10X Genomics protocol “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression” (CG000338, Rev F) for the remainder of the experiment. Input 18,000 nuclei for each tagmentation reaction for a targeted recovery of ~10,000 nuclei.