TITLE: Nuclei preparation from frozen tissue for Chromium Single Cell  Multiome ATAC + Gene Expression (10x Genomics)
AUTHORS: nzemke, Bing Ren

[DESCRIPTION]
This protocol details nuclei preparation from frozen tissue for Chromium Single Cell Multiome ATAC + Gene Expression (10x Genomics). Tissue collection for this protocol needs prior approval by the users' Institutional Review Board (IRB) or equivalent ethics committee

[STEPS]
SECTION: Nuclei preparation
1. Prechill any tubes, buffers or tools. Set the centrifuge to 4 °C.
SECTION: Nuclei preparation
2. For each sample to be homogenized, prepare a Dounce Tissue Grinder with 2 pestles (“Loose” and “Tight”). Remove from 70% ethanol storage, rinse with MilliQ water three times. Place mortars (buckets) in ice. Dry pestles with kimwipe and place on clean parafilm on top of ice to chill.
SECTION: Nuclei preparation
3. Remove excessive water collected at the bottom of mortars with p1000.
SECTION: Nuclei preparation
4. Add 990 µL of NIM-DP and 10 µL of Triton X-100 to each bucket to make 1 mL of NIM-DP-L. Pipette mix.
SECTION: Nuclei preparation
5. Tip tissue into mortar or resuspend tissue in 1 mL of NIM-DP-L buffer and transfer to a Dounce homogenizer.
 

SECTION: Nuclei preparation
6. Homogenize the sample by using the Loose pestle (usually 5-10 strokes) followed by the tight pestle (usually 15-25). Switch pestles when most of the tissue has been broken up into small pieces and use the tight pestle to homogenize until the solution is uniform (no obvious particles).
SECTION: Nuclei preparation
7. Filter using a   CellTrics filter into LoBind tube.
SECTION: Nuclei preparation
8. Centrifuge nuclei 1000 rcf, 10 min, 4 °C.

SECTION: Nuclei preparation
9. Discard the supernatant and gently resuspend pellet in 1 mL of NIM-DP. Centrifuge nuclei 1000 rcf, 10 min, 4 °C.

SECTION: Nuclei preparation
10. Add 7-AAD to Sort Buffer (1:1000) to a final concentration of 2 micromolar (µM).
SECTION: Nuclei preparation
11. Discard supernatant from pelleted nuclei. Gently resuspend pellet in 400 µL of sort buffer + 7-AAD by mixing with a p1000 pipette ~5 times or until no clumps are visible.
SECTION: Nuclei preparation
12. Sort 120,000 nuclei into a LoBind tube containing 87.5 µL of collection buffer.
SECTION: Nuclei preparation
13. Measure the total volume of the sorted nuclei in the collection buffer via reverse pipetting.
SECTION: Nuclei preparation
14. Add the appropriate amount of 5X permeabilization buffer for a final concentration of 1X, and gently pipette mix with p1000 5 times.
SECTION: Nuclei preparation
15. Incubate on ice for 1 min, then centrifuge 500 rcf, 5 min, 4 °C.

SECTION: Nuclei preparation
16. Slowly remove the supernatant until 20-30 μL remains in the tube.
 

SECTION: Nuclei preparation
17. Slowly add 650 µL of Wash Buffer along wall of tube (try not to disturb pellet) and immediately centrifuge 500 rcf, 5 min, 4 °C.
SECTION: Nuclei preparation
18. Prepare tubes for counting nuclei on hemocytometer by adding 7 µL of 1X Nuclei Buffer to each tube for a 1:16 dilution.
SECTION: Nuclei preparation
19. Very carefully remove the supernatant, switching to a p20 pipette once the supernatant volume is <20 µL. Leave ~1 µL to avoid disturbing the pellet.
SECTION: Nuclei preparation
20. Add 7 µL of 1X Nuclei Buffer and very gently resuspend pellet 4 times. Immediately take 1 µL and add to tube for counting containing 7 µL of 1X Nuclei Buffer.
SECTION: Nuclei preparation
21. Count nuclei by stain with 8 µL Trypan Blue (Invitrogen, T10282), count on a hemocytometer and record images from the microscope field. For calculating nuclei concentration of original stock multiply count average x 10 x 16 (dilution factor).
SECTION: Nuclei preparation
22. Refer to Chromium Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics) Step 1. Load 18 - 20K nuclei per tagmentation.