TITLE: Demuxlet Cell Preparation Protocol
AUTHORS:   Woong-Yang Park,   Jay Shin, Shyam  Prabhakar

[DESCRIPTION]
This protocol details Demuxlet cell preparation.

[GUIDELINES]
If cell viability for any sample is <50%, avoid using that sample in the suspension mix.
If cell viability is between 50-70%, you may try to enrich for viable cells by centrifuging at lower speed (100 g). It may improve the viability to >75%, the cells can then be mixed with the other samples.

[STEPS]
SECTION: Preparation of Reagents and Media
1. Prepare appropriate volume of thawing media (RPMI + 5% HS + 1% Pen/Strep + 1% Glutamine) and keep it at 4 °C.

SECTION: Preparation of Reagents and Media
2. Prepare appropriate volume of wash media (RPMI + 10% FBS + 1% Pen/Strep + 1% Glutamine) and and keep it at 4 °C.

SECTION: Preparation of Reagents and Media
3. Prepare appropriate volume of fresh PBS + 0.04% BSA.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
4. Warm up thawing media, wash media and PBS + 0.04% BSA in 37 °C water bath.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
5. Transfer 9 mL of 37 °C pre-warmed thawing media into each of the 15 mL Falcon tube.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
6. Take the cryovial containing PBMCs out of liquid nitrogen storage, place cryovial on dry ice, and transfer immediately to the  37 °C  water bath. Thaw for 1 minute-2 min until no visible ice crystals remain.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
7. After thawing for 1 minute-2 min, open the cryovial in a biosafety cabinet and add 500 µL-1 mL of pre-warmed thawing media into the cryovial using the 1 mL wide-bore blue tips.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
8. After adding the thawing media, use the 1 mL wide-bore blue tips to gently transfer the whole suspension from the cryovial into the 15 mL Falcon tube containing 9 mL of pre-warmed thawing media.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
9. Mix the suspension extremely gently by inverting the Falcon tube twice.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
10. Centrifuge at 300 x g, 5 min, 21 °C.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
11. Decant the supernatant.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
12. Leave around 200 µL of supernatant behind.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
13. Using a serological pipette, gently re-suspend the cell pellet (3-5 times) in 5 mL of pre-warmed wash media.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
14. Centrifuge at 300 x g, 5 min, 21 °C. 

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
15. Decant the supernatant. Leave around 200 µL of supernatant behind.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
17. Repeat Step 14-16.
Centrifuge at 300 x g, 5 min, 21 °C. Decant the supernatant. Leave around 200 µL of supernatant behind. Using a serological pipette, gently re-suspend the cell pellet (7 times) in 3 mL of pre-warmed PBS + 0.04 % BSA.
 

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
18. After the second wash, centrifuge at 300 x g for 5 min at 21 °C. Decant the supernatant until around 200 µL of supernatant is left behind.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
16. Using a serological pipette, gently re-suspend the cell pellet (7 times) in 3 mL of pre-warmed PBS + 0.04 % BSA.
 


SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
19. Gently re-suspend the cells in 800 µL of PBS + 0.04% BSA (pipette ~10 times).

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
20. Filter the cell suspension through the   Macs SmartStrainer to remove clumps or debris. After filtering, keep cells on ice.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
21. Thoroughly mix 15 µL of cell suspension with 15 µL of trypan blue. Load 10 µL of mixture into each of the two chambers of a cell counting slide.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
22. Let the samples sit in the cell counting slide for 1 min before performing cell counting on an automated cell counter.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
23. Make aliquots of 1.50 × 106 cells/mL for each sample (100 µL aliquot per sample).

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
24. Keep the remaining cell suspension from individual samples on ice for DNA extraction using a QIAamp DNA Mini Kit (Qiagen, Cat no. 51306) according to the manufacturer’s protocol.  Perform genotyping using Illumina Global Screening Array-24 v3.0 BeadChip (Cat. No.: 20030770).

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
25. Mix equal volumes (80 µL) of cell suspension from each sample (up to 16 samples) to make a pooled suspension with a final concentration of 1.50 × 106 cells/mL (total volume: 1280 µL). Keep this pooled suspension on ice.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
26. Thoroughly mix 15 µL of the pooled suspension with 15 µL of trypan blue. Load 10 µL of the mixture into each of the two chambers of a cell counting slide.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
27. Let the samples sit in the cell counting slide for 1 min before performing cell counting on an automated cell counter.

SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
28. Count cells from the pooled suspension using an automated cell counter and verify that the concentration is in the range of 1.10 x 106 cells/mL to 1.50 x 106 cells/mL.
SECTION: Thawing Frozen PMBCs and Preparing the Suspension Mix
29. Proceed with single cell capturing: Load 40,000 cells per 10x well using an appropriate 10x reagent kit for your application and run the 10x Genomics Chromium Controller according to the manufacturer’s protocol.