TITLE: Protocol for single cell and single nucleus prep for Cell Pellets/Multiple Myeloma (snRNA &amp; snATAC)
AUTHORS: Reyka Jayasinghe

[DESCRIPTION]
Protocol for single cell and single nucleus prep for Cell Pellets/Multiple Myeloma (snRNA & snATAC)

[STEPS]
SECTION: Reagents and Protocols
1. 

All lysis buffers should
be prepared fresh and stored at 4oC. 

Buffers based on Nuclei Isolation from Mouse Brain Tissue for
Single Cell ATAC Sequencing Protocol from 10X


Relevant 10X Protocols:

Nuclei Isolation for Single Cell ATAC
Sequencing
Nuclei Isolation from Mouse Brain Tissue for
Single Cell ATAC Sequencing Protocol


Chromium Single Cell ATAC Reagent Kits User
Guide (v1 Chemistry)
Chromium Single Cell 3' Reagent Kits User
Guide (v3.1 Chemistry)



SECTION: Equipment
2. •	Eppendorf DNA LoBind Tubes, 2.0 mL (022431048)
•	Tips RT-LTS-A-1000µL-/L/S/W-768/8 (30389221)
•	Flowmi® Cell Strainers, 40 μm, for 1000 uL Pipette Tips (BAH136800040)
•	Sorvall™ ST 8 Small Benchtop Centrifuge (75007204 - refrigerated)
•	Thermo Scientific™ Adapters for TX-150 Swinging Bucket Rotors (75-005-743)
SECTION: Reagents to Order for Lysis and Wash Buffers:
3. •	UltraPure™ 1 M Tris-HCI Buffer, pH 7.5 (Invitrogen, 15567027)
•	NaCl (5 M), RNase-free (Invitrogen, AM9759)
•	MgCl2 (1 M) (Invitrogen, AM9530G)
•	Nonidet P40 Substitute (Sigma, 74385-1L)
•	MACS BSA Stock Solution (Miltenyi Biotec, 130-091-376)
•	Phosphate Buffered Saline (1X) (Corning, 21-040-CM) 
•	SUPERase• In™ RNase Inhibitor (20 U/μL) (Invitrogen, AM2696)
SECTION: Buffers
4. Stock Lysis buffer: Lysis Buffer
     Reagent      Stock      Final      2 mL        Tris-HCl
  (pH 7.4)      1M      10 mM      20 mL        NaCl      5M      10 mM      4 mL        MgCl2      1M      3 mM      6 mL        Nonidet
  P40 Substitute      10%      0.1%      3 mL        Molecular
  Grade Water             -      1.974 mL   

SECTION: General Notes
10. •	Keep everything on ice.
•	Use wide-bore pipette tips for all steps when possible. 
•	Use RNase free reagents and consumables (Use filtered tips). 
•	For centrifugation steps, use swinging rotor bucket at 4°C.
SECTION: Buffers
5. Lysis Dilution Buffer
     Reagent      Stock      Final      10 mL        Tris-HCl
  (pH 7.4)      1M      10 mM      100 mL        NaCl      5M      10 mM      20 mL        MgCl2      1M      3 mM      30 mL        Molecular
  Grade Water             -      9.85 mL   

SECTION: Buffers
6. 0.1X Lysis Buffer
     Reagent      Stock      Final      10 mL        1X Lysis
  Buffer      1X      0.1X      1 mL        Lysis
  Dilution Buffer      1X      0.9X      9 mL   

SECTION: Buffers
7. Wash buffer: 1X PBS + 2% BSA + 0.2U/ul RNase inhibitor 
     Reagent      Stock       Final      10 mL        BSA      10%      2%      2 mL         PBS      1X      1X      8 mL        RNAse
  Inhibitor      20 U/mL      0.2U/ul      1 mL   



 
SECTION: Buffers
8. sn-ATAC-Seq submission
buffer: 1X Nuclei Dilution Buffer. 
We store aliquots of 20X
Nuclei Dilution Buffer at -20oC in aliquots of no more than 25ul/each
tube to minimize freeze/thaw. We make just enough fresh 1X Nuclei Dilution
Buffer each time we use it. Add 425 uL Nuclease Free Water.

SECTION: Buffers
9. Trypan blue (2X) - filtered at 0.22 mm.
SECTION: Preparing sample for auto-MACS dead cell removal
11. 1.	Add 5 mL running buffer to tube
2.	Thaw aliquot in 37⁰C water bath
3.	Spray with 70% EtOH and wipe down
4.	Add 1 mL thaw media to each aliquot and transfer to running buffer tube
5.	Remove supernatant
6.	Re-suspend each in 100 uL of beads and incubate at room temperature for 15 mins
7.	Dilute 0.25 mL of 20x binding buffer with 4.75 mL of distilled water
8.	Add 500 uL of 1x binding buffer to each tube
9.	Run each through the DepleteS selection on the Automacs
10.	Toss the positive fractions
11.	Pull off 10 uL each from negative fractions to count
12.	Proceed downstream with negative fractions, cells still suspended in buffer
SECTION: Single Cell and Nucleus Prep for sc-RNA-Seq and sn-ATAC-Seq
12. 1.	Spin down at 400 g for 5 min. Remove supernatant. 
2.	Resuspend in 1000uL Wash Buffer. 
3.	Spin down cells at 400 g for 5 min. Remove supernatant.
4.	Re-suspend in 200-1000 uL Wash buffer depending on size of the cell pellet with RNase Inhibitor. Count cells at this point – take 10uL of trypan blue and 10uL of sample and determine starting concentration of sample. If there is more than 1500 cells/uL then add more wash buffer and recount. 
5.	Aliquot cell suspension to load approximately 10,000 cells/ul (refer to 10X 3’ NEXT GEM protocol, Figure 2). Set aside another 50-100 uL of cell suspension for back up. Centrifuge remaining sample at 400g for 8’ at 4°C.
6.	While remaining cell suspension is being spun down, load 10X CHIP G (single-cell). After loading chip you have 17 minutes to process remaining sample for sn-ATAC. 
7.	After spin is finished, re-suspend cells in 500 L of cold lysis buffer. Pipette gently for 25 times (until pellet is fully resuspended). Incubate on ice for 2 min.
8.	Add 400 uL Wash Buffer to same tube.
9.	Transfer the clear lysis-wash buffer suspension using the 40um flowmi cell strainer into a 2.0 mL tube. Centrifuge sample at 400g for 5’ at 4°C.
10.	Once the sample is finished spinning, remove as much supernatant as possible. The pellet should be at the very bottom of tube if using ST8 Centrifuge. Add 7-10uL 1X Nucleus Dilution Buffer (sn-ATAC-Seq submission buffer). Gently resuspend until nuclei are completely re-suspended. Count nuclei at this point – Add 8 uL 1X Nucleus Dilution Buffer to 2 uL of sample and 10 uL of trypan blue. If concentration of nuclei is greater than 3,000 nuclei/uL then proceed with ATAC prep. 
11.	Continue both 10X protocols accordingly. 
a.	Chromium Single Cell ATAC Reagent Kits User Guide (v1 Chemistry)
b.	Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry)