TITLE: Processing PBMCs for multiplexed scRNA-seq
AUTHORS: Ning Xie, Fang Zhang, Yuanqing Feng, Sarah Tishkoff

[DESCRIPTION]
Purpose: to prepare viable single cells from frozen PBMCs for scRNA-seq, multiplexing strategy is applied to reduce batch effect and cost.

[BEFORE_START]
Carefully determine the PBMCs will be used in one experiment based on individual information. Cells from unrelated individuals can be pooled together.

[STEPS]
SECTION: Thawing PBMCs
1. Prepare Complete medium and Washing medium, and warm to 37°C in a water bath. 
Prepare 50 mL Falcon tubes with 25 mL Washing medium each.
 

SECTION: Thawing PBMCs
2. Remove cryovials from liquid nitrogen storage and place them on dry ice.

 

SECTION: Thawing PBMCs
3. Thaw frozen vials in the water bath at 37°C for 1 min (set timer), only one vial each time. Remove from the water bath when a tiny ice crystal remains. Wipe the vials with 70% ethanol and bring them to the hood.
SECTION: Thawing PBMCs
4. Pour the thawed cells gently into a 50 mL conical tube containing 25 mL pre-warmed Washing medium. Use one 50 mL conical tube for each vial. 
 

SECTION: Thawing PBMCs
5. Rinse the cryovial with 1 ml pre-warmed Washing medium and add the rinse to the 50 mL tube.
SECTION: Thawing PBMCs
6. Centrifuge at 350 xg for 5 min at room temperature. Aspirate the supernatant.
SECTION: Thawing PBMCs
7. Resuspend cell pellet in 1 mL of pre-warmed Washing medium (dropped slowly along the side of the tube) by gently tapping. Add 4 mL of pre-warmed Washing medium. Invert gently the tube several times to homogenize the cell suspension. Take 10 uL for cell counting (step 8). Add another 5 mL of pre-warmed Washing medium. Centrifuge at 350 xg for 5 min at room temperature.
SECTION: Thawing PBMCs
8. Count cells and determine viability. Count the cells by adding 10 uL of cells to 10 uL of trypan blue and pipetting up and down to mix. Apply 10 uL of the mixture to the hemacytometer or cell counter. Calculate the cell number.
SECTION: Thawing PBMCs
9. 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 350 xg for 5 min at room temperature. Note: Using Complete medium is to wash away benzonase.
SECTION: Thawing PBMCs
10. Resuspend cells in Complete medium and adjust to 1 x 10^6 cells per mL. Gently tapping.
SECTION: Thawing PBMCs
11. Seed cells into 24-well plate (low attachment plate), at 1 x 10^6 cells per well in 1 mL Complete medium, using a wide-bore 1 mL pipet tip.
SECTION: Thawing PBMCs
12. Transfer the cell culture plate to the incubator (37°C, 5% CO2) for 16-24 hrs, and leave the cells for recovery.
SECTION: Stimulation of the PBMCs with ligands
13. Prepare LPS (200ng/mL, 20x), IFNβ (2000U/mL, 20x) in Complete medium. 
 

SECTION: Stimulation of the PBMCs with ligands
14. Add 50 uL of LPS (200 ng/mL, 20x) in one well (with 1 mL medium) at a final concentration of 10 ng/mL. Or add 50 uL of IFNβ (2000U/mL, 20x) in one well (with 1 mL medium) at a final concentration of 100 U/mL. For control, add 50 uL Complete medium.
 

SECTION: Stimulation of the PBMCs with ligands
15. Tap plate 10 times (at each side of the plate) to resuspend cells and mix medium. Transfer the cell culture plate to the incubator (37°C, 5% CO2) and incubate for 6 hrs.
SECTION: Harvest PBMCs and pool samples
16. 
 

At the end of the incubation (6 hr after adding LPS or IFNβ), remove the cell culture plate from the incubator. 
Check cells under the microscope if any cell clusters. 
Transfer cells with medium into 5 mL FACS tube, using 1 mLwide-bore pipette. 
Rinse the well with 1 mL PBS and add the rinse to the FACS tube. 
Check cell plate under a microscope if any cell leftover. If cells are attached to the well, add 200 uL Tryple E, incubate at 37°C for 5 min, rinse with another 1 mL PBS and transfer to the FACS tube.
SECTION: Harvest PBMCs and pool samples
17. Centrifuge at 350 xg for 8 min at 4°C. 
Pour off the supernatant into sink (usually will have 50 uL liquid leftover). 
Invert the tube and dry the tube top with a Kim wipe.
SECTION: Harvest PBMCs and pool samples
19. Wash cells again with Staining Buffer.
Add 500 uL Staining Buffer (now total volume around 550 uL). 
Tap on the white columns to resuspend cells. 
Take 10 uL for Trypan Blue staining and count cells using cell counter Countess. 
Calculate the cell number and volumes (will use in step 21).
SECTION: Harvest PBMCs and pool samples
20. Add 2 mL Staining Buffer. 
Centrifuge at 350 xg for 5 min at 4°C. Pour off the supernatant. Dry the tube top.

 

SECTION: Harvest PBMCs and pool samples
21. Resuspend cells in the Staining Buffer and adjust to 5 x 10^6 cells per mL. 
Tap on the white columns to resuspend cells. Place cell tubes on ice.
Take out (using a wide-bore tip) the same number of cells from indicated samples and pool cells into low binding 1.5 mL Eppendorf tubes. 
 

 

SECTION: Dead cell removal
22. 
 

Filter cell with 70 um cell strainer using normal 1 mL tip. Centrifuge at 350 xg for 5 min at 4°C. Remove the supernatant.
SECTION: Dead cell removal
23. Add 100 uL dead cell removal microbeads (well-vortexed). Resuspend by pipetting using P100 pipet. Leave at room temperature for 15 min. 

 

SECTION: Dead cell removal
24. During the last 1 min, rinse the MS column with 500 uL binding buffer (room temperature). Keep MS column wet all the time. Add 400 uL binding buffer to the pooled cells. Transfer the cell suspension onto the column and leave them to pass through. Collect the flowthrough in a low binding 1.5 mL Eppendorf tube.
SECTION: Dead cell removal
25. Rinse the column with another 500 uL binding buffer. Collect the flowthrough in the same tube from the last step. Centrifuge at 350 xg for 5 min at 4°C. 
 

SECTION: Prepare single-cell suspension
26. Resuspend the cell pellet with 1 mL 0. 4% BSA in PBS, slowly pipet using a wide-bore tip. Centrifuge at 350 xg for 5 min at 4°C. Use a pipet to remove SPN.
SECTION: Prepare single-cell suspension
27. Resuspend the cell pellet with 200 uL-400 uL 0.04% BSA in PBS (depends on how big the cell pellet, if tiny pellet, do 200 uL). Pass through the 40 um Flowmi Cell Strainer using P1000. Do not spin down cells after filtration. Take 10 uL for Trypan Blue staining and count cells using cell counter Countess, record cell viability.
SECTION: Prepare single-cell suspension
28. Adjust the cell number to 1500 cells/uL (this is 10X Genomics suggestion) and place cell on ice. We will use 40 uL of 1500 cells/uL cell suspension for each reaction using 10X Genomics single cell 3' kit, i.e. 60K cells. 
SECTION: Prepare single-cell suspension
29. Move forward to 10X Genomics protocol of "Chromium Next GEM Single Cell 3' Reagent Kits v3.1" immediately.
 

SECTION: Harvest PBMCs and pool samples
18. Wash cells with Staining Buffer. 
Add 0.5 mL Staining Buffer (from BioLegend) to each sample. 
Tap on the white columns several times to resuspend cells. 
Add another 1.5 mL Staining Buffer to each sample. 
Centrifuge at 350 xg for 5 min at 4°C. 
Pour off the supernatant and dry the tube top.