TITLE: CITE-seq for PBMCs
AUTHORS: Nicola Wilson, Fernando Calero-Nieto, Javier Rodríguez-Ubreva, Bertie Gottgens, Esteban Ballestar, Roser Vento-Tormo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for CITE-seq for PBMCs. It includes the steps for Thawing samples and Antibody Staining. </div></div>

[STEPS]
?. [Thawing Samples]
Warm up 10x the volume of thawing medium (RPMI + 10% FBS).
?. [Thawing Samples]
Thaw frozen PBMCs in .
[water bath]
?. [Thawing Samples]
Transfer sample into a 15 ml tube.
?. [Thawing Samples]
Add 10x volume of thawing medium dropwise into tube.
?. [Thawing Samples]
Spray down with 70% EtOH.
?. [Thawing Samples]
Centrifuge .
Centrifuge: 500 1324, 5 min
?. [Thawing Samples]
Remove the sealed bucket from the centrifuge and place into the Class II MSC.
?. [Thawing Samples]
Discard supernatant.
?. [Thawing Samples]
Resuspend in  for  to remove erytrocytes.
[RBC lysis buffer]
?. [Thawing Samples]
Resuspend the cell pellet in .
[FACS buffer]
Equal volume for all patients.
?. [Thawing Samples]
Perform ficoll to eliminate dead cells.
?. [Thawing Samples]
Recover live cells.
?. [Thawing Samples]
Count cells using trypan blue.
One fraction of these PBMCs will be used directly and another fraction will be used for CD3+ cells depletion.
?. [Thawing Samples]
Add  and  to a centrifuge conical tube.
[Ficoll]
[cell suspension]
?. [Thawing Samples]
Centrifuge at .
Centrifuge: 10000 1324, 10 s
?. [Thawing Samples]
Swap the tube and centrifuge again at .
Centrifuge: 10000 1324, 30 s
?. [Thawing Samples]
Wash with .
[FACS buffer]
?. [Thawing Samples]
Centrifuge and discard supernatant.
?. [Thawing Samples]
Resuspend in .
[FACS buffer]
?. [Thawing Samples]
With the first fraction of PBMCs: pool 125,000 of live PBMCs from each patient (2 patients) in low binding tubes; 250,000 cells in total.
IMPORTANT: Keep the cells   all times!!
?. [Thawing Samples]
Cells should be pooled in a final volume of exactly .
100 µl
?. [Thawing Samples]
Proceed with CD3 negative cells isolation using the second fraction of PBMCs:
?. [Thawing Samples]
Centrifuge and remove . Final volume .
[supernatant]
12.5 µl
?. [Thawing Samples]
Use 2x106 of the PBMCs (  + ).
[cell suspension]
[anti-CD3 magnetic beads]
?. [Thawing Samples]
Resuspend in .
[FACS buffer]
?. [Thawing Samples]
Pass the cells through a MS magnetic column with 3 washes of .
500 µl
?. [Thawing Samples]
Centrifuge the eluted fraction and resuspend in .
[FACS buffer]
?. [Thawing Samples]
Count CD3 negative cells using trypan blue.
?. [Thawing Samples]
Pool 125,000 of CD3 negative cells from each patient (2 patients) in low binding tubes = 250,000 cells in total
IMPORTANT: Keep the cells on ice at all times!!
?. [Thawing Samples]
Cells should be pooled in a final volume of exactly .
100 µl
?. [Thawing Samples]
Incubate  at  (not ice).
4 °C
?. [Thawing Samples]
Centrifuge and remove supernatant.
?. [Thawing Samples]
Resuspend in .
[ FACS buffer]
?. [Thawing Samples]
Centrifuge and remove .
[supernatant]
Final volume  .
?. [Antibody Staining]
Prepare CITE-seq Antibody mix:
?. [Antibody Staining]
Use one aliquot of AB pool per sample; centrifuge at .
Centrifuge: 14000 1324, 1 min, 4 10
?. [Antibody Staining]
Add  to reconstitute.
[ FACS buffer]
?. [Antibody Staining]
Vortex.
?. [Antibody Staining]
Incubate in the dark for  to ensure the antibodies are completely resuspended.
?. [Antibody Staining]
Centrifuge .
Centrifuge: 14000 1324, 10 min, 4 10
?. [Antibody Staining]
During the centrifugation, add  per tube.
[Human TruStain FcX™ Fc Blocking Reagent]
?. [Antibody Staining]
Incubate  at .
4 °C
?. [Antibody Staining]
Add  to FC blocked samples, taking care to avoid any pellet (if one exists).
[CITE-seq AB mix]
?. [Antibody Staining]
Incubate for  at  in dark.
4 °C
?. [Antibody Staining]
Add .
[FACS buffer]
?. [Antibody Staining]
Place tubes in centrifuge bucket inside the Class II MSC and close the lid.
?. [Antibody Staining]
Centrifuge the samples at  (swing bucket centrifuge).
Centrifuge: 500 1324, 5 min, 4 10
?. [Antibody Staining]
Discard the supernatant.
?. [Antibody Staining]
Repeat wash steps two more times (total of 3 washes).
?. [Antibody Staining]
Resuspend cells in .
[1x PBS + 0.04 % BSA]
?. [Antibody Staining]
Filter cells through 40 μm strainers (Flowmi Cell Strainer).
?. [Antibody Staining]
Pass an extra volume of  through the same flowmi strainer to recover the remaining cells.
400 µl
?. [Antibody Staining]
Centrifuge the sample at  and remove .
Centrifuge: 500 1324, 5 min, 4 10
[supernatant]
Final volume will be around  .
?. [Antibody Staining]
Count cells using Trypan blue.
?. [Antibody Staining]
Proceed immediately to the 10x Genomics Single Cell protocol.
NB: The aim to is collect a total of ~7,000 cells per patient. For this, 25,000 cells of the pool of 2 samples will be loaded into one channel of the 10x Chromium.
?. [Antibody Staining]
Take   +  (final dilution 1:3).
[cell suspension]
[Trypan blue]
?. [Thawing Samples]
Add .
[FACs buffer]
?. [Thawing Samples]
Centrifuge at .
Centrifuge: 400 1324, 5 min
?. [Thawing Samples]
Remove supernatant.