TITLE: Nuclei isolation and permeabilisation of fresh frozen human brain samples for 10X Genomics Multiome
AUTHORS: Koen Theunis, Suresh Poovathingal, Sarah Geurs

[DESCRIPTION]
This protocol details the procedure of nuclei isolation and debris removal from fresh frozen brain tissue in preparation for the 10X Genomics multiome ATAC/GEX assay. 

[STEPS]
SECTION: Preparation
1. Prepare the 2X salt-Tris solution and aliquot to 1 ml tube and freeze at -20°C
SECTION: Preparation
2. Prepare the BSA in PBS and filter through 0.2um filter.
SECTION: Preparation
3. Prepare the Tween-20 Homogenization lysis buffer fresh for each preperations
SECTION: Preparation
4. Filter sucrose 2M solution through 0.2um/0.45um filter
SECTION: Preparation
5. Prepare cOmplete protease inhibitor (50X) by dissolving a tablet in 1 mL of water.
SECTION: Preparation
6. Filter protease inhibitor solution through 0.2um filter
SECTION: Preparation
7. Place the homogenizer at -80°C at the start and leave 10 min before homgenization on ice.
SECTION: Homogenization (Peform all steps on ice)
8. Cut brain piece on dry ice (if a large section), then transfer immediately in homogenizer containing 250 uL of TST+NP Homogenization Lysis Buffer. Immediately add 500uL TST+NP Homogenization Lysis Buffer
SECTION: Homogenization (Peform all steps on ice)
9. Allow the tissue to thaw for 2 mins in the homogenization buffer.
SECTION: Homogenization (Peform all steps on ice)
10. Homogenize tissue with pestle A (10X), wait 1min, and with pestle B (10X).
SECTION: Homogenization (Peform all steps on ice)
11. Put the homogenate through 70µm cell strainer and place the falcon on ice. Incubate on ice for 5 mins. Rinse dounce and filter with 250uL TST+NP HB.
SECTION: Homogenization (Peform all steps on ice)
12. During the incubation (in about 4 mins), use LUNA-fl counter to assess the viability of the nuclei.
SECTION: Homogenization (Peform all steps on ice)
13. Transfer the contents from the 50 mL falcon to a 2 mL protein lo-bind tube.
SECTION: Homogenization (Peform all steps on ice)
14. Centrifuge @ 500xg for 5 mins. Discard the supernatant.
SECTION: Homogenization (Peform all steps on ice)
15. Resuspend the pellet in 200 uL of Wash Buffer 1, and transfer to a 2mL DNA lobind tube.
SECTION: Homogenization (Peform all steps on ice)
16. Add additional 320 uL Wash Buffer 1 to a final volume of 520 uL.
SECTION: Homogenization (Peform all steps on ice)
17. Add 520uL of Gradient medium to sample (Vf = 1040 uL).
SECTION: Isolation by centrifugation
18. Layer 770 uL of 29% Cushion in the ultracentrifuge tube.
SECTION: Isolation by centrifugation
19. Layer the sample on top of cushion using a P1000, without disturbing the cushion.
SECTION: Isolation by centrifugation
20. Optional: Check tube weight, adjust weight to counter differences.
SECTION: Isolation by centrifugation
21. Centrifuge at least 3,000 rcf in a swinging bucket, at 4°C for 20 minutes with brake off.
SECTION: Isolation by centrifugation
22. Remove supernatant, remove the lower supernatant with P200, leaving about  50-100 uL.
SECTION: Resuspension and permeabilisation
23. Gently resuspend nuclei in ultracentrifuge tube and transfer to 1.5 mL DNA lobind tube.
SECTION: Resuspension and permeabilisation
24. Rinse ultracentrifuge tube with Wash buffer 2 and transfer also to 1.5 mL DNA lobind tube.
SECTION: Resuspension and permeabilisation
25. Centrifuge @ 350-450xg for 5 mins. Discard the supernatant.
SECTION: Resuspension and permeabilisation
26. Resuspend in 0.1x lysis buffer (200 uL) and gently pipetmix 5x
SECTION: Resuspension and permeabilisation
27. Incubate on ice for 2 min
SECTION: Resuspension and permeabilisation
28. Add Wash buffer 2 (1mL) & gently pipetmix 5x
SECTION: Resuspension and permeabilisation
29. Centrifuge @ 350-450xg for 5 mins. Discard the supernatant.
SECTION: Resuspension and permeabilisation
30. Resuspend in 1X Diluted Nuclei buffer. Count