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Can I perform cell hashing in Next GEM Universal 3’ or 5’ workflows?

Question: Can I perform cell hashing in Next GEM Universal 3’ or 5’ workflows?

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

Next GEM 5’ assays

We have performed limited in-house testing with cell hashing with Next GEM Universal 5’ v2 assays. An in-house dataset (linked here) provides an example of cell hashing with the Next GEM 5’ HT v2 assay. Cells were labeled with Biolegend TotalSeqTM-C cell hashing antibodies as well as the Biolegend TotalSeqTM-C Human Universal Cocktail. 

Next GEM 3’ assays

We have not performed in-house testing with cell hashing with Next GEM Universal 3’ v3.1 assays. 

Suggested protocols

The articles below contain suggested cell labeling protocols and suggested data analysis pipelines for cell hashing experiments:

The guidance in the above articles was based on limited testing with GEM-X Universal Singleplex 3’ v4 and 5’ v3 assays. Similar principles may apply for Next GEM assays. However, the cell loading guidance in the articles above is specific to GEM-X assay versions. The cell recovery efficiencies and the recommended maximum number of targeted cells varies between assay versions:

  • For standard throughput Next GEM 3' v3.1 and 5' v2 assays, it may be possible to target up to 30,000 cells if using cell hashing.
  • For high throughput Next GEM 3' v3.1 HT and 5' v2 HT assays, it may be possible to target up to 60,000 cells if using cell hashing.
  • The CellPlex 3’ v3.1 and CellPlex 3’ HT v3.1 User Guides provide suggested cell loading tables for recovering up to 30,000 cells and 60,000 cells respectively. 

10x Genomics has not performed extensive validation for cell hashing. We can only provide limited troubleshooting guidance. Cell Hashing is not considered fully supported by 10x Genomics. 

Products: Single Cell Immune Profiling, Single Cell Gene Expression