TITLE: 10X Multiome TST Nuclei Isolation
AUTHORS: Sébastien Vigneau, Yashika_Rustagi

[DESCRIPTION]
This protocol describes the process of nuclei isolation from frozen tissue. The protocol has been slightly modified from the Nature Medicine RNA-Seq toolbox manuscript (Slyper et al., 2020) and applied to frozen melanoma and liver metastases for the Human Tumor Atlas Network (HTAN) single-nuclei RNA-seq Trans-Network Project (snRNAseq TNP).  

[STEPS]
SECTION: PREPARATION
1. Clean off bench space with RNAse away, then 70% EtOH.
SECTION: PREPARATION
2. Set swing-bucket centrifuge to 4°C.
SECTION: PREPARATION
3. Prepare tissue culture hood by turning off UV light, opening the sash, and allowing for the 3-minute ventilation. Clean off work area with 70% EtOH and check for any supplies needed in the hood (i.e. Tips, tubes, serological tips, etc.).
SECTION: PREPARATION
4. Gather materials.
SECTION: PREPARATION
5. Clean spring scissors and forceps with 70% EtOH. Leave on Kim wipe to dry.
SECTION: PREPARATION
6. If working with OCT,prepare ice bucket with enzyme blocks and at least 50 mL PBS.
SECTION: PREPARATION
7. Take out 10x Reagents. Obtain the 20x Nuclei Buffer and ATAC Buffer from the 10x MO ATAC Kit in -20°. Obtain gel beads and Template Switch Oligo from -80°. Keep enzymes in freezer in kit until it is time to load.  
SECTION: MECHANICAL DISSOCIATION
9. Avoid thawing tissue before dissociation. If tissue is large enough for excess, cut into smaller fragments about the size of a grain of rice on ice. Note percentage of tissue used, and properly store remaining tissue in original storage case (cassette, tube, etc.) 
SECTION: MECHANICAL DISSOCIATION
10. OCT Block:
Place OCT block on dry petri dish on dry ice. Using a scalpel, scrape away thin pieces of OCT using the “parmesan cheese” method . Stop using the “parmesan cheese” method when the tissue is more visible through the OCT. Be careful not to cut away any tissue.
Add tissue to petri dish with ~at least 50 mL of cold PBS on ice (enough to submerge sample) and carefully pull away the remaining OCT using two forceps.
Transfer tissue to 1.5 mL tube on wet ice. Add 1 mL of TST buffer and chop on ice with spring scissors for 10 minutes. Note: After 3-5 minutes, suspension may be homogenized enough to instead pipette with a P1000 for the remainder of the 10 minutes.  
11. Cryoprep - (See COVARIS cryoPREP Protocol)
Transfer 1mL of TST Buffer with pulverized tissue from tissue pouch into 1.5 mL tube on wet ice. Check pulverization quality to move on to chopping or pipetting.
Chop on ice with spring scissors for 5 minutes. Note: After ~3 minutes, suspension may be homogenized enough to instead pipette with a P1000 for the remainder of the 5 minutes.   
SECTION: FILTRATION
12. Attach a 30 μm filter to a 15 mL Falcon tube on wet ice. Pass the homogenized ~1 mL nuclei suspension of nuclei through the filter.
SECTION: FILTRATION
13. Wash the previously used 1.5 mL tube with an additional 1 mL of TST buffer, then pass through the same filter.
SECTION: FILTRATION
14. Wash the filter with 3 mL of 1X ST buffer. The total volume of the suspension should be about 5 mL. Pull the suspension through the filter using a pipette if necessary.
SECTION: FILTRATION
15. Centrifuge for 10 minutes at 500 g at 4°C. Set the stop break to 5.
SECTION: FILTRATION
16. Carefully aspirate the ~5 mL of supernatant to a new 15 mL Falcon tube on wet ice. 
18. Incubate for 2 minutes on wet ice.
19. Add 1 mL of Wash Buffer and pipette mix.
20. Centrifuge for 10 minutes at 500g at 4°C.
21. Remove supernatant. Keep supernatant in new tube on wet ice.
22. Resuspend pellet in 100-150 μL of chilled Diluted Nuclei Buffer. Keep on wet ice.  
SECTION: NUCLEI QUALITY ASSESSMENT AND COUNTING
23. Count nuclei and assess quality using a hemocytometer. Dilute to desired concentration with Diluted Nuclei Buffer for easier counting, if necessary. Make a 1:2 dilution with Trypan Blue (5 μL stock, 5 μL Trypan Blue) to count.
SECTION: NUCLEI QUALITY ASSESSMENT AND COUNTING
24. To count nuclei: Count all four corners** and calculate concentration for (nuclei/μL)   

  Note: If nuclei concentration is too low for downstream workflow, nuclei can be concentrated by centrifuging for 10 minutes at 500 g at 4°C, removing a fraction of the supernatant and resuspending in the remaining supernatant.   
25. Using the table below, prepare the nuclei stock for loading.
  

27. If stock concentration is higher, create a dilution using Diluted Nuclei Buffer for optimal loading concentration.
28. If stock concentration is lower, assess volume needed to load optimal number/minimal number of nuclei. If needed, stock can be spun down and resuspended in Diluted Nuclei Buffer at a lower volume to create higher concentration for loading.
29. When creating dilution, always use at least 5 μL of nuclei stock to ensure accurate pipetting. If dilution factor is higher than 25, create a serial dilution. 
30. Once sample is prepared to be loaded, perform another quality assessment. Using a new 1.5 mL tube, add 5μL of loading sample to 5 μL of Trypan blue. Using a new hemocytometer, count the Trypan-stained sample. This calculation is to make sure the loading sample is the correct concentration. Use the calculation below to determine the concentration of the loading sample prior to loading it on the controller:

  

31. Proceed to transposition steps as outlined in “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression” User Guide (CG000338).
SECTION: Buffer Aliquot Preparation
8. Obtain aliquots from -20°C. Let thaw for approximately 20-30 minutes. Mix with P1000 once the last reagent is added. DO NOT vortex buffers. Once prepared, keep all buffers on ice. Always add Protective RNAse Inhibitor (PRI) last. DO NOT vortex PRI. See Appendix A for stock buffers. See Appendix B if making buffers the day of nuclei isolation. 
SECTION: PREPARATION
7.1. See Materials section, Preparation Materials
SECTION: Buffer Aliquot Preparation
8.2. TST
Obtain TST buffer (w/o BSA, PRI) aliquot of 1.94mL.
Prior to use add 10 μL of 20mg/mL BSA.
Add 50μL of Protective RNAse Inhibitor.
Keep on ice. 
SECTION: Buffer Aliquot Preparation
8.1. 1X ST
Obtain 1X ST buffer (w/o PRI) aliquot of 2.925mL.
Prior to use add 75 μL of Protective RNAse Inihibitor.
Keep on ice.
SECTION: Buffer Aliquot Preparation
8.3. Lysis Buffer
Obtain Lysis Buffer (w/o DTT, BSA PRI) aliquot of 874μL.
Prior to use add 1μL of DTT.
Aliquot 87.5μL into a new tube.
Add 10μL of 10% BSA to the tube from step 3.
Add 2.5μL of Protective RNAse Inhibitor to the tube from step 3.
Keep on ice.
SECTION: Buffer Aliquot Preparation
8.4. Wash Buffer
Obtain Wash Buffer (w/o DTT, BSA, PRI) aliquot of 874μL.
Prior to use add 1μL of DTT
Add 100μL of 10% BSA
Add 25μL Protector RNAse Inhibitor.
Keep on Ice.
SECTION: Buffer Aliquot Preparation
8.5. Diluted Nuclei Buffer
Obtain 20x Nuclei Buffer from 10x MO ATAC kit. Vortex and spin down the 20x Nuclei Buffer. Prepare the Diluted Nuclei Buffer using the following table: 
 

2. Keep on ice.

SECTION: NUCLEI PERMEABILIZATION
17. Resuspend nuclei pellet in 100 μL of Lysis Buffer. Pipette mix.
SECTION: LOADING THE CONTROLLER
26. Double check with Group Lead and sample sheet to determine how many nuclei to load.
29.1. Example of dilution calculation:   

Concentration of stock: 60,000 nuclei/μL 
Need a 1:12 dilution to get to 5,000 nuclei/μL 
             Create a 1:3 dilution from stock
                            5 μL of stock with 10 μL of Diluted Nuclei Buffer 
                            Left with 20,000 nuclei/μL
             From the 1:4 dilution, create a 1:4 dilution
                            5 μL of 1:3 dilution with 15 μL of Diluted Nuclei Buffer 
                            Left with 5,000 nuclei/μL
             
SECTION: Appendix A: BUFFER STOCK PREPARATION
32. Prepare the necessary buffers and solutions as outlined below for both nuclei isolation and permeabilization steps: 

SECTION: Appendix A: BUFFER STOCK PREPARATION
32.1. 2X ST Buffer Stock. (Can be stored at 4°C for up to a month.)   
  

SECTION: Appendix A: BUFFER STOCK PREPARATION
32.2. 1X ST Buffer Stock. (w/o Protective RNAse Inhibitor). 
Prep aliquots of 2.925mL.  Store at -20°C.   

  

SECTION: Appendix A: BUFFER STOCK PREPARATION
32.3. TST Buffer Stock. (w/o Protective RNAse Inhibitor and BSA). 
Prep aliquots of 1.94mL. Store at -20°C.   

  

SECTION: Appendix A: BUFFER STOCK PREPARATION
32.4. Lysis Buffer Stock. (w/o Protective RNAse Inhibitor, BSA, and DTT). 

Before preparing the remaining buffers, place the digitonin on the thermomixer at 95°C for 5 minutes at 1500 rpm. 

Dilute NP-40 substitute to a 10% solution before adding to Lysis Buffer. 

Prep aliquots of 874μL. Store at -20°C.   

  


32.5. Wash Buffer Stock. (w/o Protective RNAse Inhibitor, BSA, and DTT).

Prep aliquots of 874μL. Store at -20ºC.

   

SECTION: Appendix B: BUFFER PREPARATION
33. If no stock buffers are available, please use the following tables.
SECTION: Appendix B: BUFFER PREPARATION
33.1. 2X ST Buffer. (Can be stored at 4ºC for up to a month)
   
SECTION: Appendix B: BUFFER PREPARATION
33.2. TST Buffer. Keep on ice.

  

SECTION: Appendix B: BUFFER PREPARATION
33.3. 1x ST buffer. Keep on ice.

  
SECTION: Appendix B: BUFFER PREPARATION
33.4. Lysis Buffer.

Before preparing the 1X Lysis Buffer, place the Digitonin on the thermomixer at 95°C
for 5 minutes at 1500 rpm

Keep on ice.

  
SECTION: Appendix B: BUFFER PREPARATION
33.5. Wash Buffer. Keep on ice.
		
 

SECTION: Appendix B: BUFFER PREPARATION
33.6. Diluted Nuclei Buffer. Keep on ice.