Question: What options are available for antigen specificity studies with the Chromium Single Cell Immune Profiling assay?
Answer: This article provides suggestions for third-party reagents that may be used for T or B cell antigen specificity experiments in combination with the Chromium Single Cell Immune Profiling assay. This list is not comprehensive and additional options may be available. Not all the reagents listed below have been tested by 10x Genomics and assay performance cannot be guaranteed.
These reagents may be considered as potential alternative approaches to the 10x Genomics Barcode Enabled Antigen Mapping (BEAM) product, which will be discontinued on Dec 31st 2024.
Antigen specificity studies for T cells
1) dCODE Dextramers from Immudex (compatible with Feature Barcode technology)
- Immudex dCODE Dextramers consist of a dextran backbone with a series of antigen-presenting MHC molecules, fluorochrome molecules, and a DNA barcode that is compatible with our Immune Profiling Solution. This product can be used for determining T cell antigen specificity.
- 10x Genomics has tested and supports dCODE Dextramers with the Single Cell 5’ v2 assay. Further details are provided here: What factors should I consider when planning my antigen specificity experiment using dCODE™ Dextramer® from Immudex?
- dCODE Dextramers are expected to also be compatible with the Single Cell 5’ v3 GEM-X assay. However, 10x Genomics has not specifically tested dCODE Dextramers with the 5’ v3 assay version and therefore it is not officially supported at this time.
- See notes below regarding primer choice, library preparation and Cell Ranger analysis*
2) TotalSeq-C Streptavidin PE (compatible with Feature Barcode technology)
- Biolegend TotalSeq-C PE Streptavidin could be combined with biotinylated MHC monomers to determine T cell antigen specificity. This has not been tested by 10x Genomics and therefore is not officially supported.
- See notes below regarding primer choice, library preparation and Cell Ranger analysis*
3) Non-barcoded methods
- Cells could be labeled with peptide MHC molecules that contain a fluorophore but do not contain a DNA barcode, such as Biolegend Flex-T custom MHC tetramers. Flow sorting could be performed to isolate antigen positive cells, and these cells could be used as input into the Single Cell 5’ assay for generating Gene Expression and TCR libraries (without producing Feature Barcode libraries). This has not been tested by 10x Genomics and therefore is not officially supported.
Antigen specificity studies for B cells
1) dCODE Klickmers (compatible with Feature Barcode technology)
- dCODE Klickmers from Immudex could be combined with biotinylated antigens to determine B cell antigen specificity.
- dCODE Klickmers are theoretically compatible with Single Cell 5’ v2 and v3 assays but have not been tested by 10x Genomics and therefore are not officially supported.
- It may be beneficial to label cells with TotalSeq-C antibodies in addition to dCODE Klickmers, as is recommended for dCODE Dextramers.
- See notes below regarding primer choice, library preparation and Cell Ranger analysis*
2) TotalSeq-C Streptavidin PE (compatible with Feature Barcode technology)
- Biolegend TotalSeq-C PE Streptavidin could be combined with biotinylated antigens to determine B cell antigen specificity.
- 10x Genomics performed limited in-house tests using Biolegend TotalSeq-C PE Streptavidin instead of 10x Genomics BEAM Conjugates in the BEAM-Ab labeling protocol CG000595 with the 5'v2 assay, and found that this did not negatively impact data quality. However, this has not been extensively tested or formally validated by 10x Genomics and therefore is not officially supported. No additional guidance is available other than what is listed in this article. Testing has not been performed on the 5'v3 assay.
- For these in-house tests, the same volume of TotalSeq-C PE Streptavidin was used as is recommended for BEAM Conjugates in CG000595. TotalSeq-C-compatible primers and index plates were used for cDNA amplification and library preparation. See notes below regarding primer choice, library preparation and Cell Ranger analysis for TotalSeq-C*
- Compatibility of TotalSeq-C PE Streptavidin with the 10x Genomics Single Cell 5’ v2 assay is also demonstrated in a pre-print from Hurtado et al.
3) Non-barcoded methods
- Antigens could be conjugated to a fluorophore, using reagents such as Biolegend Streptavidin APC (catalog number 405207, see this article). Flow sorting could be performed to isolate antigen positive cells, and these cells could be used as input into the Single Cell 5’ assay for generating Gene Expression and BCR libraries (without producing Feature Barcode libraries).
* Additional notes on primer choice, library preparation and Cell Ranger analysis
1) Feature Barcode library preparation
- If products are used that have the same Feature Barcode pattern as TotalSeq-C oligos (eg. Biolegend Streptavidin TotalSeq-C, dCODE Dextramers, dCODE Klickmers), Feature Barcode libraries can be prepared using the following User Guides:
- Note that the cDNA primers and index plates required for amplification of TotalSeq-C-style olios differ from those required for amplification of BEAM Feature Barcode olios. TotalSeq-C-style oligos require Feature cDNA Primers 4 (PN 2000277) and Dual Index Kit TN Set A (PN-1000250)
2) Cell Ranger analysis for Feature Barcode libraries
- BEAM pipeline is only for BEAM libraries.
- The Cell Ranger Barcode Enabled Antigen Mapping (BEAM) pipeline, which calculates Antigen Specificity scores, can only be used with Feature Barcodes from the 10x Genomics BEAM Core kit. If alternative Feature Barcode sequences are used, the BEAM pipeline will not run. Also note that a BEAM (antigen capture) library is not compatible with GEM-X 5’ v3 chemistry in Cell Ranger pipelines. BEAM is not supported with the GEM-X 5’ v3 assay version.
- TotalSeq-C-style libraries can be run using “antibody capture” pipeline.
- If products are used that have the same Feature Barcode pattern as TotalSeq-C oligos (eg. Biolegend Streptavidin TotalSeq-C, dCODE Dextramers, dCODE Klickmers), Cell Ranger can be run if these Feature Barcode libraries are analyzed as “antibody capture” libraries. See the Cell Ranger Antibody Capture analysis page for guidance on generating Feature Reference CSV files for TotalSeq-C style Feature Barcode libraries. Running these libraries as “antibody capture” libraries will enable UMI counting of the Feature Barcodes but will not provide outputs with Antigen Specificity scores.
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)