Cell Processing – day 1

Transfer the selected cryovials from liquid nitrogen into dry ice then store in -80C. The day before seeding the plates (Day 1)

Day 1:

1. Thaw one vial of PBMCs in the water bath. Transfer immediately to 6mL of room temperature SCAIP media and mix gently using a wide-bore tip. Rinse the cryovial with an additional 1 ml of media.

2. Count cells on Countess: 10ul cells + 10ul Trypan Blue, Check for viability, and record on the Cell counting sheet page

3. Repeat for 11 additional samples.

4. Centrifuge cells at 400xg for 10 minutes.

5. Resuspend cells to 2x106 cells/mL in a culture medium.

6. Plate 4 wells x 100ul in round-bottom 96-well plates using a wide-bore tip (4wells/individual). Each individual is a separate column.

7. Incubate in SCAIP Media overnight at 37C and 5% CO2

8. For each sample, spin the remaining Cells and freeze the pellet for DNA (-80C freezer).

Cell Processing – Day 2

Protocol:

1. Prepare treatments (LPS, PHA, Dex, EtOH) – see Treatments page

2. Add 2ul of treatments to their respective wells (multi-channel).

3. Incubate for 6 hrs.

On ice:

4. Take the plate out of the incubator and pool across rows/individuals (4 pools of 12 individuals):

• Use multi-channel to gently mix the media using a wide-bore tip and transfer column1(treatment plate) to column 1 of the deep-well plate. Then repeat to transfer columns 2-12 (treatment plate) to column 1 of the deep-well plate. Next, wash columns 1-12 in the treatment plate with 100ul cold PBS then pool to column 2 of the deep-well plate.

5. Pool each row into a 5 ml tube using a wide-bore tip (4 tubes total).

6. Centrifuge @300rcf, 5 min, 4oC as per the 10X SC Protocol.

7. Remove the supernatant, wash with 5 ml ice-cold PBS+0.04% BSA, and centrifuge again.

8. Resuspend in 1ml ice-cold PBS+ 0.04% BSA.

9. If significant amounts of cell clumps or debris are observed, gently mix cells by pipetting up and down 10 – 15 times and filter cells using a Flowmi Cell Strainer (40 µm).

10. Count cells on the countess, check viability, and record.

9. Make sure cell concentration is within the target range of 0.7 M/ml – 1.2 M/ml (aim to capture 25k cells by loading 60K). Adjust if needed. Ideally, viability should be 90% and above acceptable above 80%. Proceed with the 10x Genomics® Single Cell Protocols