TITLE: CITE-Seq PBMCs with  demultiplexing
AUTHORS: Daniel V Brown

[DESCRIPTION]
Total-Seq labelling with demultiplexing
This protocol reflects a common experimental design where PBMC samples from multiple human donors are pooled together for Total-Seq (CITE-Seq) labelling. Often multiple samples from the same donor are to be profiled. An example is timepoint or treatment.

Therefore we utilise 2 levels of multiplexing
The distinct donors may be pooled and later demultiplexed using SNP genotypes. This cannot discriminate genetically identical samples.
Hashtag antibodies are used to multiplex to the timepoint or treatment

The hashtag labelling must be done separately. We setup a distinct antibody staining pool for each timepoint or treatment. 
When other extracellular markers are to be profiled with Total-Seq we make prepare these antibodies in a pool and then split the pool into aliquots, adding a different hashtag to the antibody subpools.

[BEFORE_START]
Make all preparations prior to thawing cells.
Collect ice and chill all buffers on ice.
Warm RPMI 10% FCS media in water bath
Coat 5mL FACS polysterene tubes with 2% BSA in PBS before use to minimize cell loss.

[GUIDELINES]
Although PBMCs are a very robust cell type it is advised to work as quickly as possible after beginning thawing.

[STEPS]
SECTION: Reconstituting the total-seq panel
1. The large panels come lyophilized e.g TotalSeq-C Human Universal Cocktail, V1.0
The typical staining protocol is to block 500,000 cells in 25 uL of volume (cell staining buffer + Fc blocking reagent) and then add 25 uL of the resuspended antibodies.
Total staining volume is 50uL
 

SECTION: Reconstituting the total-seq panel
1.2. Place lyophilized panel vial(s) in an empty Eppendorf tube, pulse spin to collect powder to the bottom of the tube.
SECTION: Reconstituting the total-seq panel
1.3. Add 27 µL  per tube and vortex 10 s 

SECTION: Reconstituting the total-seq panel
1.5. Transfer entire volume of all antibody cocktails to a single DNA LoBind tube = 50 µL 
 Centrifuge at 14000 x g, 10 min, 4 °C 

SECTION: Reconstituting the total-seq panel
1.6. Split the antibody cocktail tube into multiple aliquots. For example for 3 timepoints / treatments aliquot 15 µL into separate tubes.
 


SECTION: Preparing the Hashtag antibody
2. We have tested staining with  0.25 µg  of each hashtag antibody, 4x less than recommended by manufacturer which robustly labels PBMCs.
You will need a distinct hashtag antibody set for each sample.

 

SECTION: Preparing the Hashtag antibody
2.1. Centrifuge the stock tube at 10000 x g, 10 min, 4 °C  

 

SECTION: Total-seq and Hashtag staining
5. Recommended cell concentration is 22000 cells/uL  (2x107cells/mL) in staining buffer.
Concentrate pooled cell suspensions by centrifugation, leaving 15 µL 

 


SECTION: Total-seq and Hashtag staining
5.1. Aliquot 15 µL  into 2mL Eppendorf tubes.
You may have to perform 2 centrifugations to remove volume in the 10mL tube, transfer to 2mL tube then centrifuge again to reduce volume.
SECTION: Total-seq and Hashtag staining
5.2. Add 1.7 µL  Blocking reagent and mix.

SECTION: Total-seq and Hashtag staining
5.3. Incubate for 10 min 

SECTION: Total-seq and Hashtag staining
5.4. Add 16.7 µL  to the cell suspension.

SECTION: Total-seq and Hashtag staining
5.5. Incubate for 30 min 

SECTION: Snap freeze cell aliquot
5.6. While incubating take minimum 500,000 cells into a new eppendorf tube. You can use the 1.5mL used for cell counting to make this aliquot.
Pellet cells then snap freeze for the generation of bulk RNA-Seq libraries.
 

SECTION: Total-seq and Hashtag washing
5.7. Add 1.8 mL carefully resuspending cells with pipette tip and 400 x g, 5 min, 4 °C .
Use of swinging-bucket rotor is recommended for higher cell recovery.
Leave behind 100 µL 

SECTION: Total-seq and Hashtag washing
5.9. Repeat wash 2 more times   

SECTION: Total-seq and Hashtag washing
5.10. Resuspend cells in the remaining 100 µL   and combine the pools into a single 5mL FACS tube.

SECTION: Total-seq and Hashtag washing
5.11. 
If cell suspension is clumpy filter cells through 40uM Flowmi Cell Strainer or FACS (blue top) Cell Strainer. 
 

SECTION: Total-seq and Hashtag washing
5.12. Add DAPI to a concentration of 0.1 ug/mL , stock is typically 100x

SECTION: Cell sorting by FACS
6. Cells should be collected in 200uL volume in a 1.5-ml tube. 
We typically recover 2/3 cells based on number of events captured
SECTION: Cell sorting by FACS
6.1. Top up volume of FACS tube to minimum 250uL with PBS + 2% BSA
SECTION: Cell sorting by FACS
6.2. Sort viable (DAPI negative) cells into 200 µL  PBS + 2% BSA in a 1.5mL tube.
If necessary, the collected cells may be concentrated by centrifugation at 350 rcf and removing the supernatant. 
SECTION: Cell sorting by FACS
6.4. Dilute cells as necessary for appropriate input into the 10X Chromium chip.
Use PBS + 0.04% BSA to dilute.
SECTION: Capturing cells with 10x Genomics chromium controller
7. Follow the guidelines of the relevant 10x Genomics protocol
SECTION: Pooling cells for Total-Seq and hashtag staining
4. Key to experimental design is ensuring the number of cells from each sample is as similar as possible.
Refer to the cell pooling calculator.
SECTION: Prepare single-cell suspension
3. This protocol has only been used for Human Peripheral Blood Mononuclear Cells (PBMCs).
SECTION: Prepare single-cell suspension
3.1. We largely follow 10x Genomics guidelines:
https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-fresh-frozen-human-peripheral-blood-mononuclear-cells-for-single-cell-rna-sequencing

We use Benzoase in the resuspension media at 50U/mL per sample (1:5000 dilution). When washing cells normal media (without Benzoase) is used.

 

SECTION: Prepare single-cell suspension
3.2. Prior to counting resuspend in 9mL9 mL 
Be sure to thoroughly mix cell suspensions before counting
SECTION: Pooling cells for Total-Seq and hashtag staining
4.2. First prepare the pool of samples from healthy donors in a 10mL tube. This will subsequently be aliquotted into each of the staining pools. 
 


SECTION: Pooling cells for Total-Seq and hashtag staining
4.3. Next pool the experimental samples that go into the antibody staining pools. You will have one staining pool per timepoint or treatment. Use a 10mL tube.
SECTION: Pooling cells for Total-Seq and hashtag staining
4.4. Add the healthy donor pool into each antibody staining pool.
 

SECTION: Pooling cells for Total-Seq and hashtag staining
4.1. Remove 500uL of cell suspension into 1.5mL tube and count cells. Make note of cell viability (>95%) making a count of live cells and total cells (live + dead) in separate columns.

 


SECTION: Cell sorting by FACS
6.3. Count cells with manual haemocytometer
 

SECTION: Capturing cells with 10x Genomics chromium controller
7.1. 35,000 cells will be needed for each of the 10x Genomics captures
 

SECTION: Capturing cells with 10x Genomics chromium controller
7.2. 34uL is loaded on the 10x Genomics machine. Therefore a concentration of 1,029 cells/uL will need to be prepared per capture.
 

SECTION: Reconstituting the total-seq panel
1.1. Equilibrate the lyophilized panel vial(s) to room temperature for 5 minutes.


SECTION: Reconstituting the total-seq panel
1.4. Incubate 5 min 
vortex 30 s  then pulse centrifuge

SECTION: Preparing the Hashtag antibody
2.2. Add 0.5 µL of distinct hashtag antibodies to each antibody cocktail tube = 16 µL 
0.5uL * 0.5ug/uL = 0.25 µg 
 


SECTION: Capturing cells with 10x Genomics chromium controller
7.3. Follow the guidelines of the relevant 10x Genomics protocol. The samples may be left overnight in the thermocycler after the reverse transcription step.
SECTION: Total-seq and Hashtag washing
5.8. Resuspend cells in remaining 100uL and transfer to new 2mL eppendorf tube.