オリゴヌクレオチド標識抗体TotalSeq™を使用して、1細胞レベルでのタンパク質発現を解析し、ユニークな表現型を発見しましょう。これらの試薬は既存の1細胞シーケンス法に円滑に統合することができ、タンパク質とRNAを同時に解析することが可能となります。ここでは、プロテオゲノミクス解析で何ができるのか、そして、精密医療や腫瘍学、免疫学、神経科学や幹細胞研究などにおける新規アプリケーションで、TotalSeq™試薬がどのようにマルチプレックス法を可能とするのかを見てみましょう。
TotalSeq™フォーマットは、BioLegend独自の特許出願中の技術です。この技術に関して、当社との提携にご興味をお持ちの方はこちらからお問い合わせください。
TotalSeq™ oligo-conjugated antibodies enable measurement of proteins at a single-cell level and integrate seamlessly into existing single-cell RNA sequencing workflows, including Drop-Seq and those available from 10x Genomics.
Simultaneous Multiomic Data Generation: Increase the power of single-cell experiments by combining proteomic and transcriptomic data.
Reduced Dropouts: In contrast to mRNA, TotalSeq™-derived antibody tags are not highly prone to dropouts, which are essentially false negative readouts.
Ultra-High Parameter Surface and Intracellular Protein Detection: Detect hundreds of proteins in a single cell. A lower dropout rate contributes to enhanced sample clustering and the ability to better identify specific cell types.
Diverse applications: With a wide-range of mouse and human targets available, you can use TotalSeq™ antibodies in a variety of research areas, including:
Visit our 10x Genomics partnership page to learn how our TotalSeq reagents integrate with their single-cell solutions.
Access our optimized protocols for staining of surface and intracellular proteins specifically developed for use with our TotalSeq reagents for CITE-Seq applications. Explore our full list of TotalSeq antibodies for surface proteins and intracellular proteins.
For more information regarding the development of the CITE-Seq methodology, check out the CITE-Seq website.
Discover how our TotalSeq™️ reagents and recombinant proteins can be used to build antigen multimers to characterize B cell response to infection with LIBRA-seq.
Multi-Omics Resolves a Sharp Disease-State Shift between Mild and Moderate COVID-19
Su Y. et al. Cell. 2020; October. DOI: 10.1016/j.cell.2020.10.037
A major study aimed at performing multiomic analyses to characterize immune responses from COVID-19 patients. Using a panel of over 190 TotalSeq™ antibodies, this study identified novel immune cell populations that emerge in patients experiencing moderate COVID-19 severity that are expanded in severe cases. Additionally, the study identified changes within plasma metabolite profiles of patients experiencing severe COVID-19 disease that indicate that depletion of certain metabolites is correlated with the development of severe disease.
A Conserved Dendritic Cell Regulatory Program Limits Antitumour Immunity
Maier B, et al. Nature. 2020; doi: 10.1038/s41586-020-2134-y.
Immune checkpoint blockade is a recent cancer treatment method that induces a durable antitumor response. This therapy aims to mitigate the tolerogenic immune response induced by dendritic cells upon presentation of tumor antigen to T cells. However, it is only effective in a limited subset of cancer patients. This study aims to understand the mechanism by which enhancement of systemic antitumor T cell immunity occurs after neoadjuvant PD-L1 blockade. Through the use of single-cell proteogenomics, the authors identify a dendritic cell regulatory program that limits antitumor activity and is induced by expression of IL-4. Blockade of IL-4 results in increased antitumor responses, suggesting a potential treatment for the improvement of immune checkpoint therapy.
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.
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.
TotalSeq™-A | TotalSeq™-B | TotalSeq™-C | |
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10x Genomics compatibility | Single Cell Gene Expression Solution (3’, v2 and v3) and any system employing the poly(A) tail capture method | Single Cell Gene Expression Solution (3’, v3 or v3.1) with Feature Barcoding Technology and 10x Genomics Data Analysis Software1 | Single Cell Immune Profiling Solution (5’) with Feature Barcoding technology and 10x Genomics Data Analysis Software1 |
PCR handle | CCTTGGCACCCGAGAATTCCA | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNN2 | CGGAGATGTGTATAAGAGACAGNNNNNNNNNN |
Capture sequence | Poly-A [(A)30*A*A3] | NNNNNNNNNGCTTTAAGGCCGGTCCTAGC*A*A4 | NNNNNNNNNCCCATATAAGA*A*A4 |
Next-generation sequencing compatibility | Compatible with Illumina instruments | Compatible with Illumina instruments | Compatible with Illumina instruments |
Notes:
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?
Our full protocol compatible with surface protein expression is now available.
TotalSeq Format Compatibility |
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Single Cell Assay Compatibility |
Chromium Single Cell Gene Expression Flex |
BioLegend TotalSeq Intracellular Staining Protocol |
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Instrument Compatibility |
Chromium X Series |
Description |
Probe based whole transcriptome gene expression, compatible with fresh or fixed samples |
Additional Cellular Features |
Surface and Intracellular Proteins |
Multiplexing Compatibility |
TotalSeq-B Hashtags |
*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.
TotalSeq Format |
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BioLegend TotalSeq Intracellular Staining Protocol |
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10x User Guides |
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10x General Support |
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.
TotalSeq Intracellular Antibodies |
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Cocktails |
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Hashtags |
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All Products |
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.
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:
Read more about cell hashing and how they can be used to multiplex samples in the initial paper by Stoeckius M, et al.
Multiplexing samples in single-cell experiments improves sample throughout and provides cost savings. Learn from Nathan Lucas, PhD, as he describes the benefits and technical considerations for our TotalSeq™ hashtag reagents.
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.
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.
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:
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.
TotalSeq™-A Human Universal Cocktail
TotalSeq™-B Human Universal Cocktail
TotalSeq™-C Human Universal Cocktail
TotalSeq™-A Mouse Universal Cocktail
TotalSeq™-B Mouse Universal Cocktail
TotalSeq™-C Mouse Universal Cocktail
TotalSeq™-B Mouse Myeloid Cocktail, V1.0
Data shown for a subset of markers in the cocktail; download the complete dataset for all 154 primary targets and 9 isotype controls.
Human PBMCs were stained with the TotalSeq™-A Human Universal Cocktail v1.0 and processed using the 10x Genomics' Chromium Single Cell Gene Expression Solution (3’) and Illumina sequencing. Protein and RNA count data were transformed and visualized in a UMAP projection overlaid with protein and RNA expression levels. Clusters were identified based on protein expression only.
Data shown for a subset of markers in the cocktail; download the complete dataset for all 76 primary targets and 8 isotype controls.
Major immune cell lineages and selected cell states are identified using the TotalSeq™-B Mouse Myeloid Cocktail. Enzymatically dissociated mouse splenocytes were depleted of T and B cell populations, stained with the TotalSeq™-B Mouse Myeloid Cocktail and processed using the 10x Genomics Single Cell 3’ v3.1 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. Cell clusters were identified based on protein expression only.
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.
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