TITLE: PBMCs Processing for Single Cell Multiome ATAC + Gene Expression Sequencing
AUTHORS: Weiyan Jia, Etiena Basner-Tschakarjan, William Beggs, Derek Kelly, Ning Xie, Yuanqing Feng, Sarah Tishkoff

[DESCRIPTION]
This protocol was designed for the workflow of the Multiome assay on 16 PBMC samples using the 10X Next GEM Single Cell Multiome ATAC + Gene Expression Kit. We adapted Jimmy Ye Lab's pooling and demultiplexing strategy, overloading cells in each well. For the demultiplexing step using this protocol, you may need genotyping information of your samples.

[BEFORE_START]
1. This protocol is designed for sample pooling and overloading strategy and needs sample genotyping information.
2.Double-check if your samples have neutrophil contamination; if so, you should consider removing them to get good quality of your ATAC libraries. 

[STEPS]
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
1. Remove cryovials from liquid nitrogen storage and place them on dry ice.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
2. Warm The medium to 37°C in a water bath and prepare the washing Medium.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
3. Thaw frozen vials in the water bath at 37°C for 1-2 min.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
4. Pour the thawed cells gently into a 15 mL conical tube containing 10 mL pre-warmed Complete medium.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
5. Rinse the cryovial with 1 mL pre-warmed Complete medium and add the rinse to the 15 mL tube.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
6. Centrifuge at 300g for 5 min at room temperature and aspirate the supernatant.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
7. Resuspend cells with 1 mL pre-warmed Washing medium and add 9 mL more Washing medium to each tube.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
8. Incubate for 10 min at room temperature to ensure the free-floating DNA is depleted.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
9. Centrifuge at 300g for 5 min at room temperature and aspirate the supernatant.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
10. Prepare samples for Cell counting.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
10.1. Resuspend thecell pellet in 1 mL of pre-warmed Complete medium (dropped slowly along the side of the tube) by gently tapping.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
10.2. Add 4 mL of pre-warmed Complete medium.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
10.3. Invert the tube gently several times to homogenize the cell suspension.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
10.4. Take 10 uL for cell counting.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
10.5. Add another 5 mL of pre-warmed Complete medium and centrifuge at 350g for 5 min at room temperature.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
11. Count cells and determine viability.
 

SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
11.1. Count the cells by adding 10 µL of cells to 10 µL of trypan blue and pipetting up and down to mix.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
11.2. Apply 10 uL of the mixture to the hemacytometer or cell counter.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
11.3. Calculate the cell number.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
12. Resuspend cell pellet in 1 mL of pre-warmed Complete medium by gently tapping. Add another 9 mL of pre-warmed Complete medium. Briefly vortex (optional). Centrifuge at 350g for 5 min at room temperature.
 

SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
13. Resuspend cells in Complete medium and adjust to 1 x 106 cells per mL. Gently tap tubes.
SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
14. Seed cells into a 24-well plate (low attachment plate) at 1 x 106 cells per well in 1 mL Complete medium, using a wide-bore 1 mL pipet tip.
 

SECTION: 1. Thawing PBMCs( ~ 2 hrs, Benzonase treatment )
15. Transfer the cell culture plate to the incubator (37°C, 5% CO2) for 16-24 hrs, and leave the cells for recovery.
SECTION: 2. Stimulation of the PBMCs with ligands
16. Prepare LPS (200ng/mL, 20x) in Complete medium.
 

SECTION: 3. Harvest PBMCs
20. Preparation and check the following list before starting this section
 

SECTION: 2. Stimulation of the PBMCs with ligands
17. Add 50 µL of LPS (200ng/mL, 20x) in one well (with 1 mL medium) at a final concentration of 10 ng/mL.
SECTION: 2. Stimulation of the PBMCs with ligands
18. Tap the plate 10 times (at each side of the plate) to resuspend cells and mix the medium.
SECTION: 2. Stimulation of the PBMCs with ligands
19. Transfer the cell culture plate to the incubator (37°C, 5% CO2) for 6 hrs.
SECTION: 3. Harvest PBMCs
21. Move the cell culture plate out of the incubator at the end of the incubation (6 hr after adding LPS).

SECTION: 3. Harvest PBMCs
22. 350 x g, 8 min, 4 °C Centrifuge at 350g for 8 min at 4°C.

SECTION: 3. Harvest PBMCs
21.1. Check cells under the microscope to see if there are any cell clusters.
SECTION: 3. Harvest PBMCs
21.2. Transfer cells with medium into a 5 mL FACS tube using a 1 mL wide-bore pipette. Rinse the well with 1 mL PBS and add the rinse to the FACS tube.
SECTION: 3. Harvest PBMCs
21.3. Check the cell plate under a microscope to see if any cells are left over.
SECTION: 3. Harvest PBMCs
21.4. Add 200 µL Tryple E, incubate at 37°C for 5 min, and rinse with another 1 mL PBS.
SECTION: 3. Harvest PBMCs
21.5. Pipette a few times and transfer all the cells to the FACS tubes by wide-bore pipette.
SECTION: 3. Harvest PBMCs
22.1. Pour off the supernatant into the sink (usually will have 50 uL liquid leftover).
SECTION: 3. Harvest PBMCs
22.2. Invert the tube and dry the tube top with a Kim wipe.
SECTION: 3. Harvest PBMCs
23. Wash cells with Staining Buffer (from BioLegend)
SECTION: 3. Harvest PBMCs
23.1. Add 0.5 mL Staining Buffer to each sample.
SECTION: 3. Harvest PBMCs
23.2. Tap on the white columns several times to resuspend cells.
SECTION: 3. Harvest PBMCs
23.3. Add another 1.5 mL Staining Buffer to each sample.
SECTION: 3. Harvest PBMCs
23.4. Centrifuge at 350g for 5 min at 4°C.
SECTION: 3. Harvest PBMCs
23.5. Pour off the supernatant.
SECTION: 3. Harvest PBMCs
23.6. Dry the tube top.
SECTION: 3. Harvest PBMCs
24. Count cells.
SECTION: 3. Harvest PBMCs
24.1. Add 500 µL Staining Buffer (now total volume around 550 uL). Tap on the white columns to resuspend cells.
SECTION: 3. Harvest PBMCs
24.2. Take 10 µL for Trypan Blue staining and count cells using cell counter Countess.
SECTION: 3. Harvest PBMCs
24.3. Calculate the cell number and volumes (will be used in step 26).
SECTION: 3. Harvest PBMCs
25. Wash cells with Staining Buffer.
SECTION: 3. Harvest PBMCs
25.1. Add 2 mL Staining Buffer.
SECTION: 3. Harvest PBMCs
25.2. Centrifuge at 350g for 5 min at 4°C.
SECTION: 3. Harvest PBMCs
25.3. Pour off the supernatant.
SECTION: 3. Harvest PBMCs
25.4. Dry the tube top.
SECTION: 3. Harvest PBMCs
26. Pool cells
 

SECTION: 3. Harvest PBMCs
27. Place extra cells at 4°C (in case).
 

SECTION: 4. Dead cell removal(~1.5 hrs)
28. Check the list below before this section:
SECTION: 4. Dead cell removal(~1.5 hrs)
28.1. Prepare Binding buffer (from Dead cell removal kit, 4°C), and add 0.5 mL Binding buffer into 9.5 mL distilled water. Bring to RT before use.
SECTION: 4. Dead cell removal(~1.5 hrs)
28.2. Prepare 10 mL 0.4% BSA in PBS, add 400 µL 10% BSA (-20°C aliquots) into 9.6 mL PBS (no Ca2+, no Mg2+). Keep on ice.
SECTION: 4. Dead cell removal(~1.5 hrs)
28.3. Prepare 10 mL 0.04% BSA in PBS, and add 1 mL 0.4% BSA into 9 mL PBS (no Ca2+, no Mg2+). Keep on ice.
SECTION: 4. Dead cell removal(~1.5 hrs)
29. Filter cells with 70 µm cell strainer using a regular 1 mL tip. Centrifuge at 350g for 5 min at 4°C. Remove the supernatant.
SECTION: 4. Dead cell removal(~1.5 hrs)
30. Add 100 µL dead cell removal microbeads (well-vortexed). Resuspend by pipetting using P100 pipet. Leave at room temperature for 15 min.
 

SECTION: 4. Dead cell removal(~1.5 hrs)
36. Rinse the column with 500 µL binding buffer twice. Save the flowthrough in another 1.5 mL tube and place on ice (in case more cells can be collected).
SECTION: 4. Dead cell removal(~1.5 hrs)
35. Centrifuge at 350g for 5 min at 4°C and discard the supernatant.
SECTION: 4. Dead cell removal(~1.5 hrs)
34. Rinse the column with another 500 µL binding buffer. Collect the follow-through in the same tube from the last step.
SECTION: 4. Dead cell removal(~1.5 hrs)
33. Transfer the cell suspension into the column and leave them to pass through. Collect the follow-through in a low-binding 1.5 mL Eppendorf tube.
SECTION: 4. Dead cell removal(~1.5 hrs)
32. Add 400 µL binding buffer to the pooled cells.
SECTION: 4. Dead cell removal(~1.5 hrs)
31. During the last 1 min, rinse the MS column with 500 µL binding buffer (room temperature). Keep the MS column wet all the time.
SECTION: 4. Dead cell removal(~1.5 hrs)
37. Resuspend the cell pellet with 1 mL 0.4% BSA in PBS, slowly pipette using a wide-bore tip.
SECTION: 4. Dead cell removal(~1.5 hrs)
42. Centrifuge at 300g for 10min at 4 °C.
SECTION: 4. Dead cell removal(~1.5 hrs)
41. Calculate each pool's total cell number and ensure each pool has around 1M cells (this is 10x Genomics suggestion).
SECTION: 4. Dead cell removal(~1.5 hrs)
40. Take 10 µL for Trypan Blue staining and count cells using cell counter Countess, record cell viability.
SECTION: 4. Dead cell removal(~1.5 hrs)
39. Resuspend the cell pellet with 1000 µL 0.04% BSA in PBS. Pass through the 40 µm Flowmi Cell Strainer using P1000.
SECTION: 4. Dead cell removal(~1.5 hrs)
38. Centrifuge at 350g for 5 min at 4°C. Use a pipet to remove SPN.
SECTION: 4. Dead cell removal(~1.5 hrs)
43. Remove ALL the supernatant without disrupting the cell pellet and put cells ON ICE.
 

SECTION: 5. Neutrophils depletion(Optional, CD66abce MicroBead Kit, human)
44. 
 

SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
45. Add 100 µL of chilled Lysis Buffer for every 1 million cells. You need to scale up or down the volume of the lysis Buffer based on the cell numbers of each sample. Pipette mix 10 times and incubate for 3 min on ice.

 

 

SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
51. Resuspend nuclei pellet with 1000 µl chilled Wash Buffer(WSB) and pipette 5 times to mix for a total of 3 washes.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
50. Centrifuge at 500g for 5 min at 4 °C.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
49. Resuspend nuclei pellet with 1000 µl chilled Wash Buffer(WSB) and pipette 5 times to mix. There is no need for an interval timer for washing.(If the volume is more than 1.5 ml, transfer the nuclei to a 2-ml microcentrifuge tube)
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
48. Remove the supernatant without disrupting the nuclei pellet.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
47. Centrifuge at 500g for 5 min at 4 °C.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
46. Add 500 µL chilled Wash Buffer(WSB) for every 1 million cells to the lysed cells. Invert the tube 5 times to mix. You need to scale up or down the volume of the lysis Buffer based on the cell numbers of each sample.
 

SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
56. Take 2μL of nuclei suspension for counting, and record counts in the attached template.
 

SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
55. Resuspend pooled nuclei in ~50μL chilled Diluted Nuclei Buffer (DNB) to reach the target 15M/ml concentration. Maintain on ice.
 

SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
54. Remove ALL supernatant, taking care not to disturb the pellet.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
53. Centrifuge at 500g for 5 min at 4 °C.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
52. Pass nuclei suspension through a 40 µm Flowmi Cell Strainer.
 

SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
57. Proceed immediately to Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide(CG000338).
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
56.1. Make 1:5 dilution by adding 8μL DNB to 2μL nuclei.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
56.2. Count on a Countess II in duplicates.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
56.3. The desired concentration is 2.5-4M/mL for diluted nuclei and 12-20M/mL for nuclei in Transposition.
SECTION: 6 Nuclei isolation(pool cells, ~ 70 min)
56.4. If observe a clump of nuclei, filter through a 40μm strainer and count again.
SECTION: 7. Transposition( ~ 75 min)
58. During Cell count in Step 56, bring out the transposition reagents.
SECTION: 7. Transposition( ~ 75 min)
59. Based on the nuclei count from step 6.15, calculate the volume of nuclei resuspension and DNB needed to load 50k single nuclei/well to 4 wells(for 16 samples) or 6 wells (for 24 samples) using the attached template.
SECTION: 7. Transposition( ~ 75 min)
60. Refer to Single Cell Multiome User Guide for Transposition Mix and Isothermal Incubation(Page 30).
SECTION: 7. Transposition( ~ 75 min)
58.1. 
ActionItem10x PNStorageEquilibrate to RTATAC Buffer B2000193-20 °CEquilibrate to RT20X Nuclei Buffer2000207-20 °CPlace on IceATAC Enzyme B2000265/2000272-20 °CPlace on IceNuclei-Ice

SECTION: 8. GEM Generation &amp; Barcoding(~ 130 min, Move forward to 10X Single Cell Multiome protocol)
61. Prepare single-cell master mix.
SECTION: 8. GEM Generation &amp; Barcoding(~ 130 min, Move forward to 10X Single Cell Multiome protocol)
62. Bring following items to the Room 540
 

SECTION: 8. GEM Generation &amp; Barcoding(~ 130 min, Move forward to 10X Single Cell Multiome protocol)
63. Load chromium next GEM Chip J (cells are used in this step).
SECTION: 8. GEM Generation &amp; Barcoding(~ 130 min, Move forward to 10X Single Cell Multiome protocol)
64. Run the chromium controller.
SECTION: 8. GEM Generation &amp; Barcoding(~ 130 min, Move forward to 10X Single Cell Multiome protocol)
65. Transfer GEMs.
SECTION: 8. GEM Generation &amp; Barcoding(~ 130 min, Move forward to 10X Single Cell Multiome protocol)
67. Quenching Reaction.
SECTION: 8. GEM Generation &amp; Barcoding(~ 130 min, Move forward to 10X Single Cell Multiome protocol)
66. GEM incubation.
SECTION: 9. Buffers
68. Preparation before the assay:
 

SECTION: 9. Buffers
68.3. Prepare a total of 550 µl(11 vials) of RNase inhibitor.
SECTION: 9. Buffers
68.2. Cool down the centrifuge at 4 °C.
SECTION: 9. Buffers
68.1. Prepare the following buffers and keep all at 4 °C.
SECTION: 9. Buffers
69. DNB(Diluted Nuclei Buffer)
SECTION: 9. Buffers
69.1. 
1X Nuclei BufferStockFinal975μLNuclei Buffer (20X)20X1X50μLDTT1000mM1mM1μLNuclease-free Water--924μL

SECTION: 9. Buffers
69.2. 
DNBStockFinal400μL1X Nuclei Buffer1X1X390μLRNase inhibitor40U/μL1U/μL10μL

SECTION: 9. Buffers
70. WSB(Wash Buffer)
SECTION: 9. Buffers
70.1. 
WSBStockFinal2.5mL(1M)10mL(4M)15mL(6M)20mL(8M)Tris-HCl (pH 7.4)1M10mM25µL100µL150μL200μLNaCl5M10mM5µL20µL30μL40μLMgCl21M3mM7.5µL30µL45μL60μLBSA10%1%250µL1000µL1.5mL2.0mLTween-2010%0.1%25µL100µL150μL200μLDTT1000mM1mM2.5µL10µL15μL20μLRNase Inhibitor40U/μL1U/μL62.5µL250µL375μL500μLNuclease-free Water--2.1225mL8.49mL12.735mL16.98mL

SECTION: 9. Buffers
71. LSB(Lysis Buffer)
SECTION: 9. Buffers
71.1. 
LSBStockFinal0.45ml(4M)0.9ml(8M)1.8ml（16M）Tris-HCl (pH 7.4)1M10mM4.5µl9μL18μLNaCl5M10mM0.9µl1.8μL3.6μLMgCl21M3mM1.35µl2.7μL5.4μLTween-2010%0.1%4.5µl9μL18μLNP4010%0.1%4.5µl9μL18μLDigitonin5%0.01%0.9µl1.8μL3.6μLBSA10%1%45µl90μL180μLDTT1000mM1mM0.45µl0.9μL1.8μLRNase Inhibitor40U/μL1U/μL11.25µl22.5μL45μLNuclease-free Water--376.65µl753.3μL1.5066mL

SECTION: 10. Reagents Details
72. Complete medium (for PBMC culture)
 

SECTION: 10. Reagents Details
73. Washing medium
 

SECTION: 10. Reagents Details
78. Trypan blue 0.4% (filtered)
SECTION: 10. Reagents Details
77. 1X D-PBS
SECTION: 10. Reagents Details
76. 5% Digitonin: Invitrogen, BN2006.
SECTION: 10. Reagents Details
75. Lipopolysaccharide (LPS). Ultrapure lipopolysaccharide from E. coli K12 (InvivoGen, Cat. tlrl-peklps)
SECTION: 10. Reagents Details
74. Benzonase (Millipore, Cat: 71205-3)
SECTION: 10. Reagents Details
83. Bleach
SECTION: 10. Reagents Details
82. 10% BSA: MACS BSA stock solution(Miltenyi Biotec, # 130-091-376)
SECTION: 10. Reagents Details
79. Staining Buffer. Cell staining buffer (BioLegend #420201)
SECTION: 10. Reagents Details
81. Distilled water. UltraPure distilled water (Invitrogen, Cat. 10977-015)
SECTION: 10. Reagents Details
80. Dead cell removal kit (Miltenyi Biotec, Cat. 130-090-101)