TITLE: 10X-CITEseq protocol (COVID-19 patient samples +/- tetramer stain)
AUTHORS: Yang Sun, David Lee, George Hartoularos, Jimmie Ye

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Purpose</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">To detail the workflow for scCITE-Seq of 24 PBMC samples with 2 technicians. This is based on internal 10X CITEseq protocol v9.</div></div></div>

[STEPS]
?. [Antibody Reconstitution]
Take two TotalSeq-C Human Panel vials from  fridge.
4 °C
Briefly centrifuge each tube before opening.
?. [Antibody Reconstitution]
Reconstitute each vial in .
[cell staining buffer]
?. [Antibody Reconstitution]
Vortex for - or until the solution visually looks resuspended.
?. [Antibody Reconstitution]
Centrifuge at .
Centrifuge: 10000 34, 5 min, 4 10
?. [Antibody Reconstitution]
Combine antibody pool from two vials and keep at  until ready for staining.
4 °C
?. [Cell thawing]
Turn water bath on to , pre-warm .
37 °C
[cRPMI media]
?. [Cell thawing]
While cRPMI warms:
?. [Cell thawing]
Get a bucket with dry ice and a large container with regular ice.
?. [Cell thawing]
Label 24 of each of the following tube/vials:    a. 15 mL falcon tubes    b. Vicell sample tubes
?. [Cell thawing]
Bring all the vials from LN2 tank and keep on dry ice until needed.
?. [Cell thawing]
Aliquot  into each 15mL tube.
[warm cRPMI media]
?. [Cell thawing]
Thaw four vials at a time in water bath for  until only a small ice crystal remains and immediately hand over to partner to unscrew the vial aseptically.
37 °C
?. [Cell thawing]
Aliquot -  (5-6x105 cells) of thawed cells to its respective 15mL falcon tube drop by drop and mix gently twice with a P1000 pipette.
100 µl
125 µl
?. [Cell thawing]
Centrifuge at .
Centrifuge: 300 34, 5 min
During this centrifugation step, add 570µL of dPBS to each ViCell sample tube in preparation.
?. [Cell thawing]
Cap the cryovial and transfer to a Mr. Frosty and move to -80°C freezer for  before moving to LN2.
?. [Count Cells]
Add  to each ViCell sample tube if not performed previously (step 12).
[dPBS]
?. [Count Cells]
Aspirate supernatant and resuspend in , keep cells  from this step.
[cRPMI media]
on ice
?. [Count Cells]
Count cells on Vicell ( diluted in ). Record total cell count and viability, using template below:    i. If the total cell count is above 1x106, the remainder of the cells can be saved for bulk RNA-seq    ii. In ViCell counting, use cell-type PBMC and dilution factor 1:20
[sample]
[PBS]
?. [Count Cells]
Calculate volume needed to collect total 1.5 x 106 cells from all samples using the template below:    i. Calculate volume of each sample needed to get equal number of cells from each sample     ii. For samples that don’t have enough cells, thaw more cells from the same vial in -80°C freezer    iii. Make note of cell viability and also include dead cells in the total cell count. Do not use samples that have low cell viability (
?. [Bulk RNA Seq Sample Prep (optional)]
Take remaining cells from each sample after multiplexing.
?. [Bulk RNA Seq Sample Prep (optional)]
Spin at .
Centrifuge: 300 34, 5 min
?. [Bulk RNA Seq Sample Prep (optional)]
Aspirate supernatant.
?. [Bulk RNA Seq Sample Prep (optional)]
Either freeze the pellets in the -80°C or proceed to RNA extraction.
?. [Bulk RNA Seq Sample Prep (optional)]
Perform RNA Extraction as according to manufacturer’s protocol: Extract RNA with Qiagen RNeasy Plus Mini Kit (elute in 30µL) and quantify RNA with Qubit (Use 1µL for Qubit concentration).
?. [Bulk RNA Seq Sample Prep (optional)]
Use SmartSeq-2 protocol for bulk RNA library preparation.
?. [CITE-Seq protocol]
Use cell counts from ViCell to pool each sample into 5mL round bottom polystyrene tubes to get 1.5 x 106 cells in total.
?. [CITE-Seq protocol]
Centrifuge at .
Centrifuge: 300 34, 5 min, 4 10
?. [CITE-Seq protocol]
Aspirate supernatant, taking care not to disturb the cell pellet
?. [CITE-Seq protocol]
Resuspend cells in
[staining buffer]
?. [CITE-Seq protocol]
Incubate for  at .
4 °C
?. [CITE-Seq protocol]
Add  to pooled sample in FcBlock/staining buffer.
[antibody pool]
?. [CITE-Seq protocol]
Incubate for  (30-60 min recommended).
on ice
?. [CITE-Seq protocol]
Add .
[staining buffer]
?. [CITE-Seq protocol]
Centrifuge at.
Centrifuge: 300 34, 5 min, 4 10
?. [CITE-Seq protocol]
Carefully remove supernatant and save in 15mL falcon tube, taking care not to disturb the pellet.
?. [CITE-Seq protocol]
Use wide-bore pipette tip to resuspend cell pellet in .
[staining buffer]
?. [CITE-Seq protocol]
Centrifuge at .
Centrifuge: 300 34, 5 min, 4 10
?. [CITE-Seq protocol]
Carefully remove supernatant and save in the same 15mL falcon tube.
?. [CITE-Seq protocol]
Repeat (step 35-37) for one more wash, saving the supernatant:
?. [CITE-Seq protocol]
Use wide-bore pipette tip to resuspend cell pellet in .
[staining buffer]
?. [CITE-Seq protocol]
Centrifuge at .
Centrifuge: 400 34, 5 min, 4 10
?. [CITE-Seq protocol]
Carefully remove supernatant and save in the same 15mL falcon tube.
?. [CITE-Seq protocol]
Resuspend pellet in a small volume of cRPMI (ie. ; if the concentration is too high, cells can be diluted), then pass through a Flowmi strainer.
[cRPMI]
If using Flowmi strainer, be careful not to push the tip too hard as it is fragile. Note that ~ 30% of the cell suspension is lost when using a flowmi strainer.
If final volume is very small (less than   ), use conventional cell strainers
?. [CITE-Seq protocol]
Take  +  for counting with Countess and check for count and viability. (Vicell can be used here instead).
[filtered cell suspension]
[trypan blue]
a. The desired cell density is 2.5 x 106 cell/mL (or 2,500 cell/µ).b. If cells are too dense, use staining buffer to dilute cells to final density.c. Use 30.8 µL (or appropriate amount if concentration different) for each well of 10x library preparation to ensure 77,000 cells from each sample are added to the master mix.d. Note in 10X VDJ protocol, only 90µL of 100µL cell suspension+master mix is loaded to each well, so the actual cells loaded are 2,500 cell/µL*28µL*(90/100) = 50,000.e. Maximum volume of cell suspension can be loaded to 10x VDJ run is  ,make up extra volume using staining buffer.
?. [CITE-Seq protocol]
Load 70,000 cells/well into 6 wells of VDJ kits and perform GEM-RT.
?. [CITE-Seq protocol]
Save GEM-RT at  for up to 3 days or  for up to 1 week before 10X library preparation
4 °C
-20 °C
?. [CITE-Seq protocol]
Add
[Fc block reagent]
?. [CITE-Seq protocol]
Resuspend cells in
[staining buffer]
?. [CITE-Seq protocol]
Add  (#C0594)
[negative control streptavidin with biotinylated BSA]
[streptavidin]
?. [CITE-Seq protocol]
Incubate
on ice
?. [CITE-Seq protocol]
Add  (#C0591)
[S1 protein conjugated streptavidin]
[streptavidin]
?. [CITE-Seq protocol]
Incubate
on ice
?. [CITE-Seq protocol]
Add
[Fc block reagent]
?. [CITE-Seq protocol]
Incubate
on ice
?. [CITE-Seq protocol]
Add the antibody pool ( ) to pooled sample
75 µl
?. [CITE-Seq protocol]
Incubate
on ice
30-60 min recommended
?. [CITE-Seq protocol]
Add
[staining buffer]
?. [CITE-Seq protocol]
Centrifuge
Centrifuge: 300 34, 5 min, 4 10
?. [CITE-Seq protocol]
Carefully remove supernatant and save in 15mL falcon tube, taking care not to disturb the pellet
?. [CITE-Seq protocol]
Use wide-bore pipette tip to resuspend cell pellet in
[staining buffer]
?. [CITE-Seq protocol]
Centrifuge
Centrifuge: 300 34, 5 min, 4 10
?. [CITE-Seq protocol]
Carefully remove supernatant and save in the same 15mL falcon tube
?. [CITE-Seq protocol]
Repeat steps 39-41 for one more wash, saving the supernatant
?. [CITE-Seq protocol]
Use wide-bore pipette tip to resuspend cell pellet in
[staining buffer]
?. [CITE-Seq protocol]
Centrifuge
Centrifuge: 300 34, 5 min, 4 10
?. [CITE-Seq protocol]
Carefully remove supernatant and save in the same 15mL falcon tube
?. [CITE-Seq protocol]
Resuspend pellet in a small volume of resuspension buffer (i.e. 100 μL; if the concentration is too high, cells can be diluted) and pass through a Flowmi strainer
If using Flowmi strainer, be vareful not to push the tip too hard as it is fragile~30% of the cell suspension is lost when using a Flowmi strainerIf final volume is very small (less than 50 μL) use conventional cell strainers
?. [CITE-Seq protocol]
Take  and  for counting with Countess
[filtered cell suspension]
[Trypan blue]
Vicell can be used here insteadThe desired cell density is 2.5x106 cell/mL (or 2,500 cell/μL)If cells are too dense, use staining buffer to dilute cells to final densityUse 30.8 μL (or appropriate amount if concentration is different) for each well of 10x library preparation to ensure 77,000 cells from each sample are added to the mastermixNote in 10X V(D)J protocol, only 90 μL of the 100 μL cell suspension + mastermix is loaded to each well, so the actual cells loaded are 2,000 cell/μL x 30.8 μL x (90/100) = 70,000Maximum volume of cell suspension can be loaded to 10X V(D)J run is 31.7 μL, make up extra volume using staining buffer
?. [CITE-Seq protocol]
Load 70,000 cells each well into 6 wells and perform GEM-RT
?. [CITE-Seq protocol]
GEM-RT can be stored at  for up to  or at  for up to
4 °C
-20 °C