TITLE: Nuclei Preparation from Frozen Tissue for 10X Multiome using gentleMACS Homogenization and FANS, v1.2 Feb 2024
AUTHORS: ereisner

[DESCRIPTION]
This protocol describes isolation of nuclei from frozen tissue using gentleMACS homogenization 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 buffers fresh and leave on ice.
SECTION: Reagent preparation:
1.1. The volume of MACS Buffer prepared depends on the input mass of each sample. An additional 1 mL should be prepared for rinsing in addition to the homogenization volume. Refer to the chart below:
 
 

 

 

 


 

 

SECTION: Nuclei Preparation
2. Pre-chill a large, swing-bucket tabletop centrifuge to 4°C.
SECTION: Nuclei Preparation
3. Add the pre-determined homogenization volume of MACS buffer to each MACS tube on ice. 
SECTION: Nuclei Preparation
4. Immediately transfer samples to MACS tubes. If needed, resuspend tissue first with MACS buffer from the MACS tube and then transfer the full volume to the tube.
SECTION: Nuclei Preparation
5. Tighten the cap of each MACS tube and flip the tube upside down. Ensure that all tissue is submerged in the buffer.
SECTION: Nuclei Preparation
6. Thaw on ice for 1 min.

SECTION: Nuclei Preparation
7. Homogenize samples using the gentleMACS Octo Dissociator in the 4 °C cold room.
SECTION: Nuclei Preparation
8. Run the protocol “protein_01_01” for gentleMACS M tubes (~1 min).
SECTION: Nuclei Preparation
9. Quick spin M tubes to bring all liquid to the bottom of the tubes.
SECTION: Nuclei Preparation
10. Filter each sample into a 15 mL tube using a 30 um (green) Celltrics filter. 
SECTION: Nuclei Preparation
11. Rinse the sides of each MACS tube with 1 mL MACS buffer.
SECTION: Nuclei Preparation
12. Quick spin M tubes to bring all liquid to the bottom of the tubes.
SECTION: Nuclei Preparation
13. Transfer the rinse to each Celltrics filter.
SECTION: Nuclei Preparation
14. Centrifuge homogenized samples for 5 min at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
SECTION: Nuclei Preparation
15. Discard supernatants. A small volume of supernatant can be left in each tube to avoid disturbing the pellet.
SECTION: Nuclei Preparation
16. Gently resuspend each pellet in 500 µL sort buffer.
SECTION: Nuclei Preparation
17. Transfer the full sample volume to a pre-chilled 1.5 mL LoBind tube.
SECTION: Nuclei Preparation
18. Incubate on ice, protected from light, for 10 min.
SECTION: Nuclei Preparation
19. Aliquot 90 uL of 5X collection buffer into a 1.5 mL LoBind tube for each sample.
SECTION: Nuclei Preparation
20. Sort 120,000 nuclei into the 90 µL of collection buffer for each sample.
SECTION: Nuclei Preparation
21. Centrifuge for 5 min at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
SECTION: Nuclei Preparation
22. Discard supernatant.
SECTION: Nuclei Preparation
23. Gently resuspend pellet in 100 µL of NPB.
SECTION: Nuclei Preparation
24. Incubate on ice for 1 min.
SECTION: Nuclei Preparation
25. Add 900 µL of Wash buffer to each sample
SECTION: Nuclei Preparation
26. Centrifuge for 5 min at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
SECTION: Nuclei Preparation
27. Carefully discard supernatants. Switch to a P20 pipette once the volume reaches ~40 µL. Do NOT disturb the pellet. 
SECTION: Nuclei Preparation
28. Gently resuspend in 12 μL of 1X Nuclei Buffer (prepared from 10X Genomics protocol).
SECTION: Nuclei Preparation
29. Stain an aliquot of nuclei with 0.4% Trypan Blue. Load 10 μL into one chamber of a hemocytometer.
SECTION: Nuclei Preparation
30. Count nuclei in four quadrants. Average the count and determine the nuclei concentration (nuclei/μL).
SECTION: Nuclei Preparation
31. Capture images from the microscope field at 10X and 20X magnification.
SECTION: Nuclei Preparation
32. 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.