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 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:
Download our eBook: The Evolution and Future of Single-Cell Proteogenomics to learn more about applications using TotalSeq™ reagents and how they can contribute to discoveries in precision oncology.
Watch our introductory video to understand the basics behind proteogenomics-based applications.
This technology was first described by Stoeckius et al. of the NY Genome Center as they used antibodies coupled with oligonucleotides to simultaneously measure proteins and RNA at a single-cell level. They termed this method Cellular Indexing of Transcriptomes and Epitopes by sequencing or CITE-seq. Learn more about CITE-Seq and other TotalSeq™ applications in one of our webinars.
Learn more about 10x Genomics Single Cell Solutions including the Single Cell Gene Expression Solution (compatible with TotalSeq™-A and –B reagents) or the Single Cell Immune Profiling Solution (compatible with TotalSeq™-C reagents).
For more information regarding CITE-seq development and methodology, check out the CITE-seq website. Please note, we have optimized the use of our TotalSeq™ reagents using our protocols.
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.
| TotalSeq™-A | TotalSeq™-B | TotalSeq™-C | |
|---|---|---|---|
| 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) 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:
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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:
Read more about cell hashing and how they can be used to multiplex samples in the initial paper by Stoeckius M, et al.
In addition to single cell analysis in whole, 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 nuclei 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 nuclei 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 the TotalSeq™-C Human Universal Cocktail v1.0, containing 130 primary antibodies and 7 isotype control antibodies. Each antibody has been titrated using next-generation sequencing as a read-out to provide optimal discrimination of positive and negative cell populations.
The TotalSeq™-C Human Universal Cocktail v1.0 is compatible with both v1 and v2 of 10x Genomics’ single-cell immune profiling solution, allowing you to obtain single-cell multiomic data including cell surface protein expression, transcript expression, and full-length paired B cell and T cell receptor sequences.
For more information about the Universal Cocktail, view the protocol or download the antibody barcode list.
Data shown for a subset of markers in the cocktail; download the complete dataset for all 130 primary targets and 7 isotype controls.
Human PBMCs were stained with the TotalSeq™-C Human Universal Cocktail v1.0 and processed using the 10x Genomics Single Cell Immune Profiling Solution (5’) 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.
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.
