TotalSeq oligo-conjugated antibodies have been widely used to add protein data to single-cell RNA and DNA technologies. Now, the same trusted reagents, available in the new TotalSeq-Bn format, can be used to transform the 10x Genomics Visium CytAssist workflow into a multiomic spatial biology assay, with a simultaneous protein and RNA read-out from the same tissue sample.

 

TotalSeq-Bn antibodies are conjugated to an oligonucleotide containing a sequencing-compatible PCR handle, unique antibody barcode, and sequence complementary to the Visium CytAssist slide oligo pool.

 

 

 

Spatial Proteogenomics Workflow With 10x Genomics Visium CytAssist

 

 

 

The protein and gene expression libraries generated by the Visium CytAssist workflow are shown below. These libraries are analogous to the CITE-Seq libraries generated by the 10x Genomics Chromium platform for single-cell RNA and protein analysis. The main difference is that the 10x Genomics barcode unique for a single GEM is replaced by a spatial barcode that is unique for each of the thousands of spots in the Visium slide. Legacy TotalSeq-B antibodies are not compatible with Visium CytAssist.

 

 

Visium CytAssist Spatial Gene Expression – Probe-based Library

 

 

 

Visium CytAssist Spatial Protein Expression Library

 

 

 

TotalSeq™-Bn targets in the Visium Human Immune Cell Profiling Panel

 

The following TotalSeq™-Bn targets are available as part of the 10x Genomics Human Immune Cell Profiling Panel. Please contact 10x Genomics for information about the Panel and the Visium CytAssist assays.

 

Protein Marker	Description	Location	Clone	Isotype Control
Mouse IgG2a	Isotype control	Cell surface	MG2a-53	N/A
Mouse IgG1, κ	Isotype control	Cell surface	MOPC-21	N/A
Mouse IgG2b, κ	Isotype control	Cell surface	MPC-11	N/A
Rat IgG2a, κ	Isotype control	Cell surface	RTK2758	N/A
PanCK	Structural marker	Intracellular	AE-1/AE-3	Mouse IgG1, κ
PD1	Checkpoint protein	Cell surface	NAT105	Mouse IgG1, κ
PCNA	Cell proliferation marker	Nuclear	PC10	Mouse IgG2a
BCL2	Apoptotic cell marker	Mitochondrial outer membrane	100	Mouse IgG1
Vimentin	Mesenchymal cell marker	Intracellular	O91D3	Mouse IgG2a
CD8a	T cell marker	Cell surface	C8/144b	Mouse IgG1, κ
CD45RA	Functional T cell marker	Cell surface	HI100	Mouse IgG2b, κ
CD45RO	Functional T cell marker	Cell surface	UCHL1	Mouse IgG2a
CD19	B cell marker	Cell surface	R109	Rabbit IgG
PAX5	B cell marker	Nuclear	1H9	Rat IgG2a, κ
CD21	Dendritic and B cell marker	Cell surface	BU32	Mouse IgG1, κ
CD68	Macrophage marker	Cell surface	BL13756	Mouse IgG1, κ
CD163	Macrophage marker	Cell surface	QA19A16	Mouse IgG1, κ
HLA-DR	Myeloid cell marker	Cell surface	LN3	Mouse IgG2b, κ
CD66b	Neutrophil marker	Cell surface	6/40c	Mouse IgG1, κ
CD138	Plasma cell marker	Cell surface	DL-101	Mouse IgG1, κ

 

 

TotalSeq™-Bn Single Catalog Products

 

TotalSeq™-Bn single conjugate antibodies can be supplemented into 10x Genomics Human Immune Cell Profiling Panel. This offers the ability to expand the number of targets that can be interrogated by the Visium CytAssist Spatial Gene and Protein Expression assay. In order to optimize the concentration(s) and pool the additional antibody marker(s), refer to 10x Genomics Custom Add-on Antibody Optimization guide. For more information regarding the TotalSeq™-Bn single antibodies, please reach out to your local BioLegend Field Application Scientist or BioLegend’s Technical Service team.

 

 

Compatibility Information
10x Genomics	Visium CytAssist Spatial Gene and Protein Solution with Capture Sequence technology (FFPE) and 10x Genomics Data Analysis Software (Space Ranger and Loupe Browser)
Next-generation sequencing	Compatible with Illumina instruments
Tissue	FFPE tissue
Slide capture area	6.5 mm2 or 11 mm2

 

 

The TotalSeq™-Bn format is not compatible with 10x Genomics Chromium single-cell solutions and CITE-Seq applications. More information about TotalSeq™ in single-cell applications and CITE-Seq can be found here.

 

 

Explore Additional Resources

 

Learn more about the Visium spatial solution:

• Visium Spatial Gene and Protein Expression
• Visium data analysis pipeline (Space Ranger)
• Visium data visualization (Loupe Browser)
• 10x Genomics Custom Add-on Antibody Optimization guide