Aug 02, 2023
WU sn-prep Protocol for solid tumors- ATAC v2.7
- 1Department of Medicine, Washington University in St. Louis, St. Louis, MO 63110, USA
Protocol Citation: Wagma Caravan, Reyka Jayasinghe, Nataly Naser Al Deen 2023. WU sn-prep Protocol for solid tumors- ATAC v2.7. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdkx2lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 01, 2023
Last Modified: August 02, 2023
Protocol Integer ID: 85814
Abstract
Single nuclei dissociation protocol for snATAC-seq
Reagents and Tools
Reagents and Tools
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
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
0.1x Lysis Buffer (10mL):
1x Lysis Buffer (1mL) + Lysis Dilution Buffer (9mL)
Wash and Resuspension buffer (WR buffer, 10mL):
1X PBS, 8mL
10% Stock BSA Solution (MACS, 130-091-376), 2mL
Trypan blue (2X) - filtered at 0.22μm
7-AAD (7-Aminoactinomycin D) (Millipore Sigma SML1633-1ML)
Glass homogenizers (Fisher: 2mL tube- K8853030002, Small clearance pestle- K8853020002, Large clearance pestle- K8853010002)
Fine forceps and scalpels
General Notes
General Notes
- Keep everything on ice (or in the cold room)
- Avoid foam and bubbles as much as possible with gentle strokes and pipetting slowly
- Use RNase/DNase free reagents and consumables
Nuclei Dissociation
Nuclei Dissociation
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, 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.
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, 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.
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, 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.
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.
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).
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, 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).
Add 50μL 10% BSA/PBS solution into a 2mL nonbinding tube for collection. Sort 300-400K of the nuclei into the collection tube.
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).
Resuspend with 3μL 2x Nuclei Dilution Buffer (prepared as directed by 10x Genomics), final volume should be ~10μL.
Quantify nuclei quality and quantity using a hemacytometer or Countess II utilizing Trypan blue.
Load desired concentration of nuclei, proceed with the protocol as outlined by 10x Genomics.