Can I perform Cell Hashing in the 5' workflow?

Question: Can I perform Cell Hashing in the 5' workflow?

 

Answer: Although cell hashing is not officially supported by 10x Genomics, we have developed additional resources to help enable these experiments to improve customer success.

 

TotalSeq™-C antibodies can be used for Cell Hashing in the 5' workflow, and can be purchased from BioLegend directly.  We strongly recommend that you reach out directly to BioLegend technical support (tech@Biolegend.com) for recommendation for antibodies, etc prior to initiating your experiments.   Titrating TotalSeq™ antibodies prior to initiating applications like CITE-seq is critical for optimal results.  If the concentration of the hashing antibody is too high, it may overpower the other antibody reads and thus other more lower abundant antibodies may be under represented. 

 

Please review these 10x resources:

• 10x Demonstrated Protocol: Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols
• 10x Technical Note: Quality Control of Cell Surface Protein Labeling using Flow Cytometry
• Analysis Guide: Demultiplexing and Analyzing 5’ Immune Profiling Libraries Pooled with Hashtags
• Published Dataset: Demultiplexing 5’ Immune Profiling Libraries Pooled with Hashtags

General Recommendations for Sample Quality:

• Perform pilot experiments to determine if the sample type is suitable for the cell hashing.
• Use single cell suspensions with >80% (ideally >90%) viability. If one or more samples in the pool has lower viability, fluorescence activated cell sorting (FACS) after labeling may increase viability.

To enable cell hashing, the Demonstrated Protocol for Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols must be modified.  The required changes are described below.  The recommended number of cells for this modified protocol is 0.2-2 x 10^6 cells per sample. 

 

Cell Surface Protein Labeling Protocol modifications (page 6)

Prepare Antibody Mix Supernatant

• Add appropriate/manufacturer's recommended amount of antibody-oligonucleotide conjugates to a 1.5-ml microcentrifuge tube
• TotalSeq™ antibodies including hashing antibodies (View cell hashing products) should be titrated to determine the optimal labeling concentration prior to use. Please review the following Technical Note for additional information: Quality Control of Cell Surface Protein Labeling using Flow Cytometry.
• TotalSeq™pre-pooled antibody panels, such as the TotalSeq™ Universal cocktails are  lyophilized and need reconstitution before proceeding with staining cells.
• Equilibrate the lyophilized panel vial to room temperature for 5 minutes.
• Rehydrate lyophilized panel in PBS + 10% FBS using appropriate reconstitution volume needed for the number cells 
•  For  0.2-2 x 10^6 cells per sample**, add 25-50ul of PBS + 10% FBS to the lyophilized panel. Vortex and incubate for 10min at room temperature. Then, centrifuge the vial at 14000 rcf for 10min at 4°C.
• Transfer the supernatant (containing Antibody Mix) to a low-bind tube and maintain at 4°C.
• When performing dual staining with cell hashing antibodies and TotalSeq™ antibodies, we recommend adding cell hashing antibodies into each respective sample’s TotalSeq™ antibody pool. If sequentially staining samples, stain with cell hashing antibodies first and then pool your hashtag labeled samples for TotalSeq™ antibody staining. Please note that in some cases cell recovery may be diminished.
• The recommended amount of hashing antibody is 0.1-0.25 μg per sample.  Add appropriate volume of the hashing antibody to the Antibody Mix and maintain at 4°C.
• Make sure that each sample is stained using an Antibody Mix containing a different hashing antibody, before pooling the samples.  Please see the illustration below:

HTO - Hashtag Oligo, CSP- Cell Surface Protein

 

Cell Surface Protein Labeling protocol (starting at page 6, step d):

1. Resuspend cell pellet for each sample in 50 μl chilled PBS + 10% FBS. 
2. Add 5 μl Human TruStain FcX to each sample. Gently pipette mix.
3. Incubate for 10 min at 4°C.
4. Add the prepared Antibody Mix supernatant (TotalSeq™ cell surface antibodies + hashing antibody) to each sample as shown in illustration above.  Each sample should be stained with a different hashing antibody before pooling the samples
5. Add chilled PBS + 1% BSA to the cells to bring the total volume to 100 μl. Gently pipette mix 10x (pipette set to 90 μl). For samples containing <70% viable cells, add chilled PBS + 10% FBS.
6. Incubate for 30 min at 4°C. If using FACS antibodies, incubate without light exposure. 

 

Recommended Wash Protocol: Protocol 2

In Brief, samples should be washed three times, by adding 3.5 ml chilled PBS + 1% BSA to the labeled cells. After each wash, the resuspended cell pellet or cells should be transferred to a new 5-ml tube.  Centrifugation speed and time depends upon the sample type. Use Table 2 for guidance.  Please see Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols  for additional details. 

 

• OPTIONAL: For enrichment of labeled and viable cells by FACS: 
  • Based on starting concentration and assuming ~50% cell loss, add an appropriate volume chilled PBS + 2% FBS (including a dead cell marker) to obtain a final cell concentration of 5-10 x 10^6 cells/ ml and proceed to FACS (see FACS Guidance in Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols  for additional details).
  • After FACS, determine cell concentration and viability.
• If not performing FACS:
  • Based on starting concentration and assuming ~50% cell loss, add an appropriate volume chilled PBS + 1% BSA to obtain a concentration of 700- 1,200 cells/μl. 
  • Samples should be slowly filtered using 40 µm Flowmi™ Cell Strainer.

 

Sample Multiplexing for Cell Hashing:

• For optimal multiplet detection and optimal signal-to-noise ratios, we recommend pooling samples at 1:1 ratios.
• Determine cell concentration and viability of the pooled sample. Proceed immediately to relevant Chromium Single Cell Immune Profiling protocols with Feature Barcode technology: Select User Guide.
• Ideally the viability should be >90% after filtration for optimal capture rate. The presence of a large number of non-viable cells can decrease the efficiency of cell partitioning and recovery within the 10x Genomics Chromium chip.

 

Recommended maximum number of cells that can be profiled when using modified protocol for cell hashing:

In each Standard 10x Single Cell Immune Profiling library, it is possible to target up to 30,000 cells if using Cell Hashing. In each High-throughput 10X Single Cell Immune Profiling library, it is possible to target up to 60,000 cells if using Cell Hashing. 

Exceeding the maximum number of recommended cells per library may increase the undetected multiplet rate and risk for microfluidic failures (i.e. wetting or clogging failures). For additional information, please see: What is the maximum number of cells that can be profiled?

 

Sequencing:

We recommend a sequencing depth of a minimum 5,000 read pairs/cell for each Feature Barcode library for obtaining sufficient coverage.  If sequencing Single Cell 5' v2 or v3 Dual Index Cell Surface Protein libraries with 5’ GEX and VDJ libraries, see: What sequencing configuration should I use?.  If sequencing Single Cell 5' v2 Dual Index Cell Surface Protein libraries independently, they may also be sequenced in a 26 x 10 x 10 x 25 bp configuration. If sequencing Single Cell 5' v3 Dual Index Cell Surface Protein libraries independently, they should be sequenced in a 28 x 10 x 10 x 25 bp configuration.

 

For additional information please see:

• Sequencing Requirements for Single Cell V(D)J
• Chromium Single Cell V(D)J Libraries – Sequencing Metrics for Illumina® Sequencers

 

Products: Single Cell Immune Profiling