Question: What is the difference between TotalSeq A, B, and C?
Answer: BioLegend offers three different formats of antibody-oligonucleotide conjugates that are compatible with 10x Genomics applications as part of its TotalSeq™️ line. These antibody formats are TotalSeq™️ A, TotalSeq™️ B, and TotalSeq™️ C. Each format has a different sequence used for capturing the antibody barcode GEM generation.
- TotalSeq™️ A is compatible with any Single Cell RNA-seq product that uses poly(dT) for mRNA capture. This format can be used for CITE-seq and REAP-seq applications. As this product requires reagent and protocol modifications, 10x Genomics provides only limited support for TotalSeq™️ A. Please contact New York Genome Center or BioLegend for additional assistance with the assay or software.
- TotalSeq™️ B is specifically designed to be compatible with the Single Cell 3’ v3.1 workflow and is fully supported by 10x Genomics. TotalSeq™️ B antibodies are also compatible with the Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) workflow for Singleplexed samples. It contains a capture sequence ("Capture Sequence 1") complementary to the capture sequence on the Single Cell 3' v3.1 and Flex Gel Bead oligos. To generate Protein libraries from TotalSeq™️ B antibodies please refer to the appropriate User Guide for either the Single Cell 3’ v3.1 or Single Cell Flex assay.
TotalSeq™️ C is specifically designed to be compatible with the Single Cell 5' workflow and is fully supported by 10x Genomics. It contains a capture sequence complementary to the Template Switch Oligo (TSO) on the Single Cell 5' Gel Bead oligos. To generate Cell Surface Protein libraries from TotalSeq™️ C antibodies, please see Chromium Single Cell V(D)J Reagent Kits with Feature Barcoding technology for Cell Surface Protein.
TotalSeq™️ C antibodies are also compatible with Single Cell Gene Expression Flex for Singleplexed and Multiplexed samples via Barcode Oligo Capture. Please see: What Fixed RNA Profiling applications are compatible with TotalSeq™️ antibodies? and What does “Barcode Oligo Capture” mean in the context of Feature Barcode Technology with Fixed RNA Profiling? To generate Protein Expression libraries from TotalSeq™️ C antibodies with the Single Cell Flex assay please see the appropriate User Guide for either Singleplexed or Multiplexed samples.
Note: Cell hashing using TotalSeq™️ B or TotalSeq™️ C is not officially supported by 10x Genomics, more details can be found in Can I perform Cell Hashing in the 5' workflow? Cell Ranger 3.0 or higher and Loupe Cell Browser 3.0 or higher are required for analyzing Cell Surface Protein libraries.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex