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: Contain a poly (A) capture sequence that mimics a natural mRNA. Designed to work with platforms that utilize poly (dT) oligonucleotides as the mRNA capture method. Compatible with 10x Genomics 3' Universal Gene Expression assay, captured via the poly (dT) sequence of the Gel Bead primers. Use of TotalSeq-A reagents with this assay requires reagent and protocol modifications; 10x Genomics provides only limited support for TotalSeq-A. For additional information regarding the use of TotalSeq-A reagents, please refer to our protocols or contact BioLegend Technical Support.

TotalSeq™-B: The capture sequence is compatible with the Capture Sequence 1, or Partial Capture Sequence 1 of the barcoded Gel Bead primers used with the 10x Genomics 3’ Universal Gene Expression or Flex Gene Expression assays, respectively. Use of TotalSeq-B antibodies with these assays is fully supported by 10x Genomics.

TotalSeq™-C: The capture sequence is compatible with the Template Switch Oligo Capture Sequence, or the Partial Capture Sequence 1 of the barcoded Gel Bead primers used with the 10x Genomics 5' Universal Gene Expression or Flex Gene Expression assays, respectively. The use of TotalSeq-C antibodies with these assays is fully supported by 10x Genomics.

For more information regarding compatibility of our TotalSeq products with partner solutions, such as 10x Genomics, please visit our Technology Partnerships and Service Providers page.

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 3' Universal Gene Expression assay, additional reagents for preparation of libraries must be purchased. Please refer to our protocols for a complete list.

Notes:

• N represents a randomly selected A, C, G, or T.
• The symbol * indicates a phosphorothioated bond, to prevent nuclease degradation.
• TotalSeq™-A,-B, and -C, antibodies 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 and -C Reagents for Intracellular and Cell Surface Protein Staining

Intracellular proteins contribute to a host of cellular 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 cell surface, cytoplasmic, and nuclear proteins using TotalSeq antibodies. To preserve antigen integrity and epitope accessibility in different cellular compartments -cytoplasmic and nuclear- we have developed two distinct protocols. Each protocol uses a specialized buffer system optimized to ensure robust protein staining while maintaining a high-quality RNA signature when paired with the 10x Genomics Flex Gene Expression assay.

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 protocols for detection of cytoplasmic or nuclear proteins can be found below.

TotalSeq™ Format Compatibility

Assay Compatibility

*Use of BioLegend’s intracellular staining protocols and cell hashing reagents with 10x Genomics Chromium Single Cell Gene Expression Flex assay is not officially supported by 10x Genomics. For additional information regarding use of TotalSeq-B and -C intracellular antibodies or TotalSeq Hashtags please contact BioLegend Technical Services.

Workflows

TotalSeq™-B or -C Cell Surface and Cytoplasmic Protein Staining with 10x Chromium Single Cell Gene Expression Flex

TotalSeq™-B or -C Cell Surface and Nuclear Protein Staining with 10x Chromium Single Cell Gene Expression Flex

Required User Guides and Support

Important:

*TotalSeq-B products are only compatible with 10x Genomics Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples With Feature Barcode technology for Protein.

**TotalSeq-C products can be used with GEM-X v1 and v2 Flex Gene Expression Reagent Kits for Singleplex or Multiplex samples with Feature Barcode technology for Protein Expression, and Next GEM Fixed RNA Profiling Reagent Kits for Singleplexed or Multiplexed Samples with Feature Barcode technology for Protein using Barcode Oligo.

For additional support in choosing the correct Flex Gene Expression user guide to pair with our TotalSeq products please contact BioLegend Technical Services.

TotalSeq Reagents

TotalSeq-B: The capture sequence is compatible with the Capture Sequence 1, or Partial Capture Sequence 1 of the barcoded Gel Bead primers used with the 10x Genomics 3' Universal Gene Expression or Flex Gene Expression assays, respectively. Use of TotalSeq-B antibodies with these assays is fully supported by 10x Genomics.

TotalSeq-C: The capture sequence is compatible with the Template Switch Oligo Capture Sequence, or the Partial Capture Sequence 1 of the barcoded Gel Bead primers used with the 10x Genomics 5' Universal Gene Expression or Flex Gene Expression assays, respectively. The use of TotalSeq-C antibodies with these assays is fully supported by 10x Genomics.

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 Single-Cell Multiomic Workflows with TotalSeq™ Antibody Cocktails

Gain flexible cellular phenotyping with our TotalSeq™ antibody cocktails. These pre-defined cocktails integrate seamlessly with various single-cell RNA sequencing workflows, allowing you to analyze from nine to hundreds of proteins simultaneously, depending on your experimental goals. Each cocktail is expertly designed with pre-titrated amounts of lyophilized antibodies to optimize your workflow.

Benefits of using antibody cocktails:

• Provided in convenient, single-use tubes. 
• Antibodies are pre-titrated and balanced using next-generation sequencing as the readout for optimal performance.
• Reduces inconsistences associated with pipetting and handling multiple vials and samples. 
• Lower cost compared to buying individual antibodies. 
• Minimizes variability between different experiments, labs, and longitudinal studies. 

Quick Selection Guide

TotalSeq™ Cocktail Descriptions