Demuxlet Cell Preparation Protocol

Prepare appropriate volume of thawing media (RPMI + 5% HS + 1% Pen/Strep + 1% Glutamine) and keep it at 4°C.

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

Prepare appropriate volume of fresh PBS + 0.04% BSA.

Warm up thawing media, wash media and PBS + 0.04% BSA in 37°C water bath.

Transfer 9mL of 37°C pre-warmed thawing media into each of the 15 mL Falcon tube.

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-0h 2m 0s until no visible ice crystals remain.

After thawing for 1 minute-0h 2m 0s, open the cryovial in a biosafety cabinet and add 500µL-1mL of pre-warmed thawing media into the cryovial using the 1mL wide-bore blue tips.

After adding the thawing media, use the 1mL wide-bore blue tips to gently transfer the whole suspension from the cryovial into the 15 mL Falcon tube containing 9mL of pre-warmed thawing media.

Mix the suspension extremely gently by inverting the Falcon tube twice.

Centrifuge at 300x g,21°C.

Decant the supernatant.

Leave around 200µL of supernatant behind.

Using a serological pipette, gently re-suspend the cell pellet (3-5 times) in 5mL of pre-warmed wash media.

Centrifuge at 300x g,21°C.

Decant the supernatant. Leave around 200µL of supernatant behind.

Using a serological pipette, gently re-suspend the cell pellet (7 times) in 3mL of pre-warmed PBS + 0.04 % BSA.

Repeat Step 14-16.

Centrifuge at 300x g,21°C. Decant the supernatant. Leave around 200µL of supernatant behind. Using a serological pipette, gently re-suspend the cell pellet (7 times) in 3mL of pre-warmed PBS + 0.04 % BSA.

After the second wash, centrifuge at 300x g,0h 0m 0s for 0h 5m 0s at 21°C. Decant the supernatant until around 200µL of supernatant is left behind.

Gently re-suspend the cells in 800µL of PBS + 0.04% BSA (pipette ~10 times).

Filter the cell suspension through the 30 µm Macs SmartStrainer to remove clumps or debris. After filtering, keep cells On ice.

Thoroughly mix 15µL of cell suspension with 15µL of trypan blue. Load 10µL of mixture into each of the two chambers of a cell counting slide.

Let the samples sit in the cell counting slide for 0h 1m 0s before performing cell counting on an automated cell counter.

Make aliquots of 1.50 × 10⁶ cells/mL for each sample (100µL aliquot per sample).

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).

Mix equal volumes (80µL) 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.

Thoroughly mix 15µL of the pooled suspension with 15µL of trypan blue. Load 10µL of the mixture into each of the two chambers of a cell counting slide.

Let the samples sit in the cell counting slide for 0h 1m 0s before performing cell counting on an automated cell counter.

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.

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.