TITLE: WU sn-prep Protocol for solid tumors- joint snRNA+ATAC v2.9
AUTHORS: Reyka Jayasinghe, Wagma Caravan, Andrew Houston, Nataly Naser Al Deen

[DESCRIPTION]
Nuclei dissociation protocol adapted from WU sn-prep Protocol for Solid Tumors - snRNA protocol v2.8 for simultaneous profiling of genetic expression (snRNA) and chromatin accessibility (snATAC)



[STEPS]
SECTION: Reagents and Tools
1. 1x Lysis buffer (2mL):
10mM Tris-HCl (pH 7.4) (Thermo; 15567027), 20μL
10mM NaCl (Thermo; AM9759), 4μL
3 mM MgCl2 (Thermo; AM9530G), 6μL
NP-40 substitute (Sigma, 74385-1L), 2μL
1 M DTT (Sigma, 646563), 2μL
Nuclease Free Water (Invitrogen, AM9937), 1.966mL
SECTION: Reagents and Tools
3. 0.1x Lysis Buffer (10mL):
1x Lysis Buffer (1mL) + Lysis Dilution Buffer (9mL)
SECTION: Reagents and Tools
4. Wash and Resuspension buffer (WR buffer, 10mL):
1X PBS, 8mL
10% Stock BSA Solution (MACS, 130-091-376), 2mL
SECTION: Reagents and Tools
6. Trypan blue (2X) - filtered at 0.22μm
SECTION: Nuclei Dissociation
11. If using frozen tissue sample, use a scalpel (aided by a pair of fine forceps) to cut the cold samples (25-35mg) into 2mm pieces, add 1 ml of ice-cold 0.1X lysis buffer and 30 μL RNase Inhibitor, load into the glass homogenizer. Homogenize with 4-6 push/pulls using the pestel, incubate on ice for 1 min with an additional 500-1000μL of 0.1X cold lysis buffer. Pipette gently for 4 times. Incubate on ice again for up to 1 min.
SECTION: General Notes
10. Keep everything on ice (or in the cold room)
Use RNase free reagents and consumables, before starting wipe down all surfaces/pipettes with RNase away and 70% ethanol 
Avoid foam and bubbles as much as possible with gentle strokes and pipetting slowly
SECTION: Reagents and Tools
7. 7-AAD (7-Aminoactinomycin D) (Millipore Sigma SML1633-1ML)
SECTION: Reagents and Tools
8. Glass homogenizers (Fisher: 2mL tube- K8853030002, Small clearance pestle- K8853020002, Large clearance pestle- K8853010002)
SECTION: Reagents and Tools
9. Fine forceps and scalpels
SECTION: Reagents and Tools
5. Roche Protector RNase inhibitor-2000 U/μL (Millipore Sigma 3335399001)
SECTION: Nuclei Dissociation
11.1. If using pulverized powder, start with 15-35 mg total, add 1 ml of ice-cold 0.1X lysis buffer and 30 μL RNase Inhibitor, pipette the powder/lysis buffer mix gently for 6-8 times. Let sit on ice for 30”. Pipette another 4-6 times with an additional 500-1000μL of 0.1X cold lysis buffer. Incubate on ice again for 1’ – could be reduced (to like 20-45”). Add to the glass homogenizer, use the pestle for 3-4 push/pulls.
SECTION: Nuclei Dissociation
11.2. If using OCT sections, start with 300-450μm total sectioned into a 1.5mL tube. Add 1 ml of ice-cold 0.1X lysis buffer and 30 μL RNase Inhibitor, pipette the mix gently for 10-12 times, will be sticky as the OCT thaws. Let sit on ice for 30”. Pipette another 4-6 times with an additional 500-1000μL of 0.1X cold lysis buffer. Incubate on ice again for 1’ – could be reduced (to like 20-45”). Add to the glass homogenizer, dounce with the pestle for 6-8 push/pulls.
SECTION: Nuclei Dissociation
12. Filter the homogenate through a 40μM cell strainer on ice on top of a 50ml conical tube. Wash the filter with 1ml WR buffer. Collect this into the same tube, the total filtrate is ~3 ml. 
If there is still tissue on the strainer, backwash with 2 mL 0.1x Lysis buffer, follow previous steps again to dissociate completely. If going to FACS with the backwash, proceed with this sample as if it were a different tissue but sort into same collection tube.
SECTION: Nuclei Dissociation
13. Transfer the filtrate to a 5ml Eppendorf tube. Centrifuge at 500g for 6’ at 4°C, resuspend with 100-400μL WR buffer (depending on pellet size) and 10 μL RNase inhibitor.
SECTION: Nuclei Dissociation
14. Transfer into a FACS sorting tube and add 3μL 7-AAD per 500 μL of resuspended sample, incubate for 10 minutes before sorting at FACS. After resuspending sample in wash buffer, if small chunks are still visible (after ~3 minutes of resuspension) use 40μM mini-strainer over FACS tube to remove chunks (proceeding to FACS with sample in current condition will clog machine and will result in additional lost sample).
SECTION: Nuclei Dissociation
15. Add 50μL 10% BSA solution and 10μL RNase inhibitor and into a 2mL nonbinding tube for collection. Sort 300-400K of the nuclei into the collection tube. 
SECTION: Nuclei Dissociation
16. After FACS sorting is done centrifuge the 2ml collection tube at 500g for 6’ at 4°C. There will not be a visible pellet, remove all the supernatant (likely ~3μL will be left).
17. Resuspend in 3μL 2x Nuclei Dilution Buffer (prepared as directed by 10x Genomics, pg. 28) and 1μL RNase inhibitor, final volume should be ~10μL.
18. Quantify nuclei quality and quantity using a hemacytometer or Countess II utilizing Trypan blue. 
19. Load desired concentration of nuclei, proceed with the protocol as outlined by 10x Genomics which can be found here.
SECTION: Reagents and Tools
2. Lysis Dilution Buffer (10mL):
10mM Tris-HCl (pH 7.4) (Thermo; 15567027), 100μL
10mM NaCl (Thermo; AM9759), 20μL
3 mM MgCl2 (Thermo; AM9530G), 30μL
1 M DTT (Sigma, 646563), 10μL
Nuclease Free Water (Invitrogen, AM9937), 9.840mL