CITE-Seq PBMCs with demultiplexing
Daniel V Brown
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This protocol has only been tested on PBMCs and constituent immune subsets.
This protocol contains deviations from standard 10x Genomics SOPs and will not be supported by them.
Abstract
Total-Seq labelling with demultiplexingThis 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.
Steps
Reconstituting the total-seq panel
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
Equilibrate the lyophilized panel vial(s) to room temperature for 5 minutes.
Place lyophilized panel vial(s) in an empty Eppendorf tube, pulse spin to collect powder to the bottom of the tube.
Add 27µL per tube and vortex 0h 0m 10s
Incubate 0h 5m 0s
vortex 0h 0m 30s then pulse centrifuge
Transfer entire volume of all antibody cocktails to a single DNA LoBind tube = 50µL
Centrifuge at 14000x g,4°C
Split the antibody cocktail tube into multiple aliquots. For example for 3 timepoints / treatments aliquot 15µL into separate tubes.
Preparing the Hashtag antibody
We have tested staining with 0.25µg 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.
Centrifuge the stock tube at 10000x g,4°C
Add 0.5µL of distinct hashtag antibodies to each antibody cocktail tube = 16µL
0.5uL * 0.5ug/uL = 0.25µg
Prepare single-cell suspension
This protocol has only been used for Human Peripheral Blood Mononuclear Cells (PBMCs).
We largely follow 10x Genomics guidelines:
We use Benzoase in the resuspension media at 50U/mL per sample (1:5000 dilution). When washing cells normal media (without Benzoase) is used.
Prior to counting resuspend in 9mL9mL
Be sure to thoroughly mix cell suspensions before counting
Pooling cells for Total-Seq and hashtag staining
Key to experimental design is ensuring the number of cells from each sample is as similar as possible.
Refer to the cell pooling calculator.
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.
First prepare the pool of samples from healthy donors in a 10mL tube. This will subsequently be aliquotted into each of the staining pools.
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.
Add the healthy donor pool into each antibody staining pool.
Total-seq and Hashtag staining
Recommended cell concentration is 22000cells/uL (2x107cells/mL) in staining buffer.
Concentrate pooled cell suspensions by centrifugation, leaving 15µL
Aliquot 15µL 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.
Total-seq and Hashtag washing
Resuspend cells in the remaining 100µL and combine the pools into a single 5mL FACS tube.
If cell suspension is clumpy filter cells through 40uM Flowmi Cell Strainer or FACS (blue top) Cell Strainer.
Add DAPI to a concentration of 0.1ug/mL , stock is typically 100x
Add 1.7µL Blocking reagent and mix.
Incubate for 0h 10m 0s
Add 16.7µL to the cell suspension.
Incubate for 0h 30m 0s
Snap freeze cell aliquot
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.
Add 1.8mL carefully resuspending cells with pipette tip and 400x g,4°C .
Use of swinging-bucket rotor is recommended for higher cell recovery.
Leave behind 100µL
Resuspend cells in remaining 100uL and transfer to new 2mL eppendorf tube.
Repeat wash 2 more times
Cell sorting by FACS
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
Top up volume of FACS tube to minimum 250uL with PBS + 2% BSA
Sort viable (DAPI negative) cells into 200µL 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.
Count cells with manual haemocytometer
Dilute cells as necessary for appropriate input into the 10X Chromium chip.
Use PBS + 0.04% BSA to dilute.
Capturing cells with 10x Genomics chromium controller
Follow the guidelines of the relevant 10x Genomics protocol
35,000 cells will be needed for each of the 10x Genomics captures
34uL is loaded on the 10x Genomics machine. Therefore a concentration of 1,029 cells/uL will need to be prepared per capture.
Follow the guidelines of the relevant 10x Genomics protocol. The samples may be left overnight in the thermocycler after the reverse transcription step.
