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.

Currently, we offer TotalSeq™-A, -B, and –C formats and continue to work with compatible partners to support new versions of genomics reagents and solutions. All TotalSeq™ products are guaranteed until their expiration date (a minimum of 4 months from the date of receipt).

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 both oligos derived from 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.

Clustering of approximately 5,000 CITE-seq single-cell expression profiles of PBMCs reveals distinct cell populations based on transcriptome analysis. The left panel shows a two-dimensional representation (tSNE) of global gene expression relationships among all cells. Major cell types in peripheral blood can be discerned based on marker gene expression as indicated. The right panels show mRNA (blue) and corresponding Antibody-Derived Tag (ADT, green) signal for the CITE-seq antibody panel projected on the tSNE plot. Darker shading corresponds to higher levels measured.

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.

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.

Note: Our TotalSeq™-A TBNK panel (Cat# 399901) panel has been optimized on human PBMCs. Using lysed whole blood and additional TotalSeq™ antibodies may require further optimization. Please contact our technical service group for further information.

Remove the need for optimization and minimize variability with our pre-defined TotalSeq™ antibody panels. Each single-use tube contains a pre-titrated amount of lyophilized antibodies.

Benefits of using TotalSeq™ Panels:

• Provided in convenient, single-use tubes.
• Pre-titrated using optimal concentrations for staining.
• Reduced cost compared to buying individual antibodies.
• Minimize variability between experiments, between different labs, and during longitudinal studies.

Human PBMCs were stained with the TotalSeq™ Human TBNK panel containing TotalSeq™-A antibodies against CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD45, and CD56 (NCAM).

Download and view antibody barcode information for all of our TotalSeq™ reagents.  Check the boxes next to each antibody in your panel to export barcode information for only your antibodies of interest.  Or, select download as excel file to download information for all of our products.