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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the preparation of PBMC cells.</div></div>

[STEPS]
?. [Preparation of Reagents and Media]
Prepare appropriate amount of thawing media including RPMI + 5% HS + 1% Pen/strep + 1% glutamine and keep it for a maximum of 2 weeks at .
4 °C
?. [Preparation of Reagents and Media]
Prepare appropriate amount of wash media including RPMI + 10% FBS + 1% Pen/strep + 1% glutamine.
?. [Preparation of Reagents and Media]
Prepare appropriate amount of PBS + 0.04% BSA.
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Warm up thawing media, wash media and PBS (Ca- Mg-) at .
37 °C
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Transfer  of  warm thawing media into a .
9 mL
37 °C
[falcon tube]
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Take the tube with PBMCs out of Liquid Nitrogen and transfer on dry ice immediately to the  water bath and thaw for  to .
37 °C
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
After  to , open the tube under the laminar hood and add  (it’s a dropwise addition) into the tube using the wide-bore blue tips.
[warm thawing media]
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Having added the warm media, transfer the whole suspension into the  which contains  using the wide-bore blue tips.
[falcon tube]
[warm thawing media]
It should be added very gently (dropwise or while you keep the tube at 45 degree).
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Mix the solution extremely gently by pipetting (3-4 times) using the wide-bore blue tips (without any bubbling).
The cells at this stage are very fragile.
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Centrifuge at .
Centrifuge: 300 33, 5 min
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Re-suspend the cells in  very gently.
[pre-warmed wash media]
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Centrifuge at .
Centrifuge: 200 33, 5 min
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Repeat Step 14 and 15.
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
After the second wash, re-suspend the cells in  .
[PBS + 0.04% BSA]
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Filter the cells through the 30-50 µm cell strainer (FACS tubes) to get rid of clumps or debris.
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Count the cells and make aliquots of 1x106 cells/mL (500 µL).
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Mix equal volumes () of cell-suspension from each sample (up to 16 samples) to make a mixed suspension with a final concentration of 1x106 cells/mL which will be used for 10X single cell capture.
100 µl
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Count cell number in the mixed suspension again and keep it  for further processing of single cell capturing using 10X Chromium.
on ice
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Decant the supernatant.
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Re-suspend the cell pellet very gently by pipetting (5-10 times) using the wide-bore blue tips.
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Keep the rest of the cells at  for DNA extraction and genotyping with Illumina Global Screening Array-24 v3.0 BeadChip (Cat. No.: 20030770) according to the manufacturer’s protocol.
-20 °C
?. [Thawing Frozen PMBCs and Preparing the Suspension Mix]
Decant the supernatant and re-suspend the cells in
[pre-warmed PBS + 0.04% BSA.]