Discover Antibody-Oligo Formats

Each TotalSeq™ antibody is conjugated to a unique oligonucleotide containing a capture sequence, a clone-specific barcode sequence, and a PCR handle compatible with Illumina® sequencing reagents and instruments. Barcodes are placed between the PCR handle and 3’ flanking sequence. The oligonucleotide sequence that is conjugated to our TotalSeq™ antibodies is also referred to as an Antibody-Derived Tag, or ADT. The oligo sequence that is conjugated to our hashtag reagents is referred to as a Hashtag Oligonucleotide, or HTO.

For single-cell protein and RNA analysis, we offer TotalSeq™-A, -B, and –C formats and continue to work with compatible partners to support new versions of genomics reagents and solutions. 

Understand the Differences Between TotalSeq™ Formats

TotalSeq™-A: Designed to work with any sequencing platform that relies on poly(dT) oligonucleotides as the mRNA capture method.  TotalSeq™-A antibodies contain a poly(A) sequence which mimics a natural mRNA.

TotalSeq™-B: Capture sequence is compatible with 10x Genomics Chromium Single Cell Expression Solution 3’ kit with Feature Barcode Technology (v3 or v3.1).

TotalSeq™-C: Capture sequence is compatible with the 10x Genomics Chromium Single Cell Immune Profiling Solution (5’) which allows for immune repertoire profiling of T and B cell receptors at a single-cell resolution.

10x Genomics' Solutions, compatible with TotalSeq™-B and –C  antibodies, contain all necessary reagents to process and amplify oligos derived from both TotalSeq™ reagents and mRNA. When using TotalSeq™-A reagents with the 10x Genomics' Single Cell Gene Expression Solution Kits (v2, v3, v3.1), additional reagents for preparation of libraries must be purchased. Please refer to our protocols for a complete list.

Notes:

1. 10x Genomics' Data Analysis Software does not support cell hashing analysis.
2. N represents a randomly selected A, C, G, or T.
3. The symbol * indicates a phosphorothioated bond, to prevent nuclease degradation.
4. These sequences are unique to the TotalSeq™-B and -C conjugates, and were developed independently of the reagents used by researchers at the New York Genome Center (NYGC, CITE-seq.com). TotalSeq™-B, -C, and the antibodies used by NYGC are all compatible with 10x Genomics' Solutions, but utilize different oligo sequences. As such, protocols and additional reagents, including required primers, differ between the antibody formats. Please refer to our protocols when using TotalSeq™ reagents.

TotalSeq™-B Reagents for Intracellular Cytoplasmic and Cell Surface Staining

Intracellular proteins contribute to a host of cellar functions including cell growth, gene regulation, chemotaxis, and immunomodulation. Measuring both intracellular and surface proteins at the single-cell level can give researchers a comprehensive understanding of the cell and the molecular processes that underlie cellular function.

The ability to detect cell surface proteins with gene expression in single-cell analysis has been well established using CITE-Seq. However, detecting intracellular proteins while preserving RNA quality for gene expression profiling has proven challenging, due largely to the conditions utilized to access the intracellular compartment of cells which can have deleterious effects on RNA quality. 

With advancements in single-cell RNA technology and workflow optimization, it is now possible to observe all 3 modalities using TotalSeq antibodies and our TotalSeq™-B or -C Cell Surface and Intracellular Cytoplasmic Protein Staining with 10x Chromium Single Cell Gene Expression Flex.

Why add intracellular targets to your single-cell experiment? 

• Improve cell characterization by identifying functional molecules. 
• Add depth to cell state identification by understanding signaling pathways. 
• Discover unique and new phenotypes within seemingly homogeneous cell populations. 

Our full protocol compatible with surface protein expression is now available.

TotalSeq™ Format Compatibility

Assay Compatibility

 *Use of  the TotalSeq-B Cell Surface and Intracellular Cytoplasmic Protein Staining with 10x Chromium Single Cell Gene Expression Flex Protocol and our cell hashing reagents with 10x Single Cell Gene Expression Flex is not officially supported by 10x Genomics. For additional information regarding use of TotalSeq-B or TotalSeq Hashtags please contact BioLegend Technical Support.

Workflow

Required User Guides and Support

TotalSeq Reagents

TotalSeq-B: Capture sequence is compatible with capture sequence 1 found on the Single Cell 3' v3.1 Gel Beads used with the 10x Genomics Chromium Single Cell Gene Expression solution and 10x Genomics Single Cell Gene Expression Flex solution with Cell Surface Protein Labeling.

Additional TotalSeq Resources

TotalSeq Resources and Learning - View all our TotalSeq resources including training videos, protocols, and app notes.

Barcode Look-Up - Access our barcode look-up table to view and export barcode information for all of our TotalSeq reagents.

Intracellular and Surface Staining Representative Data

Multiplex Samples with Hashtag Antibodies

Cell Hashing

To pool multiple samples prior to loading them onto a platform capable of single-cell isolation, try one of our hashtag reagents. Each pre-mixed, ready-to-use hashtag reagent contains a pool of antibodies designed to recognize ubiquitously expressed cell surface markers and is conjugated to a unique barcode. For human samples, our hashtag reagents recognize CD298 and β2-Microglobulin, and for mouse samples, hashtag antibodies recognize CD45 and H-2 MHC Class I.

Benefits of using cell hashing:

• Robust multiplet identification.
• Minimize variability and reduce batch effects between samples.
• Pool smaller samples to reach minimal cell numbers.

Read more about cell hashing and how they can be used to multiplex samples in the initial paper by Stoeckius M, et al.

Nuclei Hashing

In addition to single-cell analysis in whole and intact cells, single-nucleus analysis makes it possible to characterize cellular states and physiology in tissues that are challenging to dissociate. This includes tissues that are rich in certain cell types like neurons, adipocytes and muscle cells. Single-nucleus analysis can also help when tissue storage is required, as it is difficult to recover and obtain single-cell suspensions from archived frozen material.

To pool isolated nuclei from different samples, we developed nuclear hashing reagents. Our nuclear hashtag antibodies recognize a family of nuclear pore complex proteins and can cross-react with vertebrate organisms and other invertebrate species, such as Xenopus and yeast.

Read more about single nucleus hashing analysis in a Nature Communications paper first describing the technology by Gaublomme, et al.

Understand the Differences between Hashtag Formats

We offer hashtag reagents in our TotalSeq™-A, B, and –C formats. Hashtag reagents from all formats will function similarly, but the workflow and required primers differ.

For TotalSeq™-A reagents, the PCR handle between antibodies and hashtag reagents are different. In practice, this means that you will generate two separate libraries– one for your cell surface markers (referred to as ADTs) and one for hashtag-derived oligos (referred to as HTOs). This is beneficial because the two libraries can be mixed at different ratios prior to sequencing to avoid an excess of reads from the hashtags.

For TotalSeq™-B and –C reagents, the PCR handle is the same between both your antibodies of interest and the hashtag antibodies. In practice, this means that the hashtag and antibody libraries must be prepared together. To avoid an excess of HTO readings, we recommend titrating down the hashtag reagents. For more information on how to titrate TotalSeq™ products, read our FAQs.

Simplify the Proteogenomics Workflow With Antibody Cocktails

Remove the need for additional optimization and minimize variability between experiments using pre-defined TotalSeq™ antibody cocktails. Each single-use tube contains pre-titrated amounts of each lyophilized antibody.

Benefits of Using TotalSeq™ Cocktails:

• Provided in convenient, single-use tubes
• Pre-titrated antibodies for optimal performance
• Offer reduced cost when compared to buying individual antibodies
• Minimize variability between different experiments, different labs, and during longitudinal studies

Characterize Cell Diversity to an Unprecedented Level with Universal Cocktails

Take a deeper look at immune cells with our TotalSeq™ Human and Mouse Universal Cocktails. Each cocktail contains over 95 antibodies and their associated isotype controls for large-scale protein detection. Within the cocktail, each antibody has been individually titrated using next-generation sequencing as a read-out to provide optimal discrimination of positive and negative cell populations.

Human Universal Cocktails

Identify Major Immune Cell Types with TBNK Cocktails

Our TBNK Panels are designed to identify T, B, and NK cells as defined by the expression of CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD45, and CD56. The TBNK cocktail is available in TotalSeq™-A, B, or C formats and is compatible with a variety of single-cell platforms.

Each cocktail has been optimized using human PBMCs. Using lysed whole blood or additional TotalSeq™ antibodies may require further optimizations. Please contact our technical service group for guidance.

Human PBMCs were stained with the TotalSeq™ Human TBNK panel containing antibodies against CD3, CD4, CD8, CD19, CD56, CD14, CD11c, CD16, and processed using the 10x Genomics' Single Cell 3’ v3 feature barcoding kit and Illumina sequencing. Protein count data were transformed and visualized in a UMAP projection overlaid with protein expression levels for each component of the cocktail. Clusters were identified based on protein expression only.