TITLE: Nuclei isolation from fresh frozen human colon tissue for 10X Genomics Multi-omics (ATAC + GEX) assay
AUTHORS: Antonina Mikorska, Koen Theunis, Suresh Poovathingal, sam kint, Sarah Geurs, Thierry Voet

[DESCRIPTION]
Nuclei isolation protocol used for sn multi-omics assay on fresh frozen human colon tissue (full thickness and muscularis layer only).

[STEPS]
SECTION: Sample preparation
1. Place the sample preparation instruments on dry ice: biopsy punch, 1.5 mL EP tube, tweezers, petri dish. 
SECTION: Experiment preparation
3. Prepare all buffers fresh and on wet ice. Add the the detergents, DTT and RNase inhibitor just before use.
SECTION: Tissue homogenization
5. Perform all steps on wet ice. Add 500 µL Collagenase Lysis Buffer to the tube with multiplexed patient samples. Let thaw slightly.
SECTION: Nuclei permeabilization
19. Resuspend the pellet in 500 µL 0.1x Lysis Buffer, pipette mix X5 with P1000.
SECTION: Nuclei resuspension and counting
23. Resuspend the pellet in 25-50 µL 1X Diluted Nuclei Buffer. 
SECTION: Sample preparation
2. Using 2 mm biopsy punch, aliquot 1.5-2 tissue pieces per patient. Place the pieces in a fresh 1.5 mL EP tube placed on dry ice. Repeat with a fresh biopsy punch for each multiplexed patient. 

SECTION: Tissue homogenization
6. Cut the tissue with scissors until there are no more pieces visible. Time = around 4 min (depending on the amount and the characteristics of the tissue). 
SECTION: Tissue homogenization
7. Further homogenize the tissue using an automatic plastic pellet pestle, for 45 s.
SECTION: Tissue homogenization
11. Wash the pestle with 250µL Collagenase Lysis Buffer and add it to the tissue.
SECTION: Tissue homogenization
10. Homogenize the tissue with the used pellet pestle x15 (by hand).
SECTION: Tissue homogenization
9. Wash the scissors using 250 µL Collagenase Lysis Buffer (while adding it to the tissue). 
SECTION: Tissue homogenization
8. Mince the tissue for 1 min. 
SECTION: Tissue homogenization
6.1. After cutting, place the scissors in a fresh 1.5 mL EP tube placed on ice.
SECTION: Tissue homogenization
7.1. Place the pellet pestle in the EP tube instead of the scissors. 
SECTION: Tissue homogenization
12. Gently pipet up and down 10X with P1000.
SECTION: Tissue homogenization
13. Centrifugate for 5 min at 500g at 4°C, remove the supernatant and resuspend in 1 mL Wash Buffer 1.
SECTION: Tissue homogenization
14. With P1000, transfer the sample onto 40 and 20 µm stacked strainers placed on 25 mL EP tube. 
SECTION: Experiment preparation
4. Place all the plastics and filters on wet ice.
SECTION: Tissue homogenization
15. Wash the 1.5 mL EP tube with additional 1 mL Wash buffer 1, transfer onto the top filter.
SECTION: Tissue homogenization
16. Centrifugate the 25 mL EP tube for 5 min at 500g, 4°C.
SECTION: Tissue homogenization
17. Resuspend the pellet in 700 µL of Wash Buffer 1 and filtrate through Flowmi filter into a fresh DNA LoBind 1.5 mL EP tube.
SECTION: Tissue homogenization
16.1. Gently remove the supernatant.
SECTION: Tissue homogenization
18. Centrifugate for 5 min at 500g at 4°C
SECTION: Tissue homogenization
18.1. Gently remove the supernatant.
SECTION: Nuclei permeabilization
21. Add 1000 µL of Wash Buffer 2 and pipette mix 5X with P1000.
SECTION: Nuclei permeabilization
20. Incubate on ice for 1 min (tissue quality dependent).
SECTION: Nuclei permeabilization
22. Centrifugate for 5 min at 500 g at 5°C. 
SECTION: Nuclei permeabilization
22.1. Very gently remove the supernatant.
SECTION: Nuclei resuspension and counting
24. Cell count the nuclei and proceed to the 10X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression assay.