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TCR profiling View data: human single-cell TCR profiling
TCR profiling View data: human bulk TCR profiling

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TCR profiling View data: human single-cell TCR profiling
TCR profiling View data: human bulk TCR profiling

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T-cell receptor profiling

Cellular immunity is mediated by T cells, which participate directly in the detection and neutralization of pathogenic threats via T-cell receptors (TCRs). Given the relative specificity of TCR-antigen interactions, a high diversity of TCRs are required to recognize a myriad of pathogenic agents. To this end, the adaptive immune system has evolved a mechanism for somatic diversification of TCRs that is unrivaled in all of biology. TCRs are heterodimers composed of two distinct subunit chains, most commonly α and β chains, resulting from somatic rearrangements in a process called V(D)J recombination (Figure 1). An extensive repertoire of T cells with diverse TCR sequences is critical for the immune system to recognize mutated proteins and neoantigens from tumor cells. One challenge of immunogenomics applied to cancer is to determine T cell repertoires and to identify tumor-reactive T-cell clones, before and in response to immunotherapy in cancer patients (Liu et al. 2017).

T-cell receptor structure and diversification

Figure 1. T-cell receptor structure and diversification. Panel A. A functional αβ TCR heterodimer consisting of α- and β-subunit chains. TCR-α subunit chains are comprised of "variable" (V), "joining" (J), and "constant" (C) segments depicted in magenta, blue, and green, respectively, while TCR-β subunit chains include these and an additional "diversity" (D) segment, depicted in orange. The CDR3 region of the TCR-β subunit is labeled. The TCR is represented on the T-cell surface, bound to an antigen associated with an MHC molecule on the surface of an APC. Panel B. V(D)J recombination and posttranscriptional processing of a TCR-β subunit chain. The TCR-β locus includes over 50 V segments (magenta), 2 D segments (orange), and 13 J segments (blue). During somatic diversification, at least one of each segment type is randomly selected, and further variability is introduced through the incorporation and deletion of additional nucleotides (yellow). Splicing of TCR mRNA combines a subset of the respective segments (along with a constant region) into a continuous unit. TCR-α subunit chains are generated via analogous mechanisms.

Highlighted products

SMARTer immune profiling kits leverage our SMART technology and a 5'-RACE-based approach to capture full-length information from V(D)J variable regions of TCRs. These kits streamline the process of sample preparation, provide reproducible results for a wide range of inputs of mouse/human samples (purified cells, spleen, PBMCs, Jurkat cells, etc.), and are highly sensitive in detecting low-abundance transcripts. SMARTer human/mouse TCR a/b profiling kits are the ideal tools for TCR profiling to gain insights into TCR repertoire diversity from bulk samples (total RNA or purified cells). Since the unique alpha-beta chain pairing of a TCR mediates antigen specificity, obtaining pairing information is crucial to gaining insights into antigen recognition, designing TCRs for targeted immunotherapy, and establishing ancestral relationships of T-cell populations (Wegehaupt et al. 2017).

The SMARTer Human scTCR a/b Profiling Kit enables the full capture of TCR-α and TCR-β variable regions to elucidate TCR α/β pairing information within single T-cells. In addition, the ICELL8 Human TCR a/b Profiling Reagent Kit for can capture complete V(D)J variable regions of TCR transcripts from >1,000 single cells.


References:

Liu, X. S. & Mardis, E. R. Applications of immunogenomics to cancer. Cell 168, 600–612 (2017).

Wegehaupt, A. K. et al. Recovery and assessment of leukocytes from LR Express filters. Biologicals 49, 15–22 (2017).


Featured products

Cat. # Product Size Price License Quantity Details
634431 SMARTer® Human scTCR a/b Profiling Kit 96 Rxns USD $4052.00

License Statement

ID Number  
325 Patent pending. For further information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The SMARTer Human TCR a/b Profiling Kit enables users to analyze T-cell receptor (TCR) diversity from single T cells that have been sorted into a 96-well plate. As the name suggests, the kit can be used to generate data for both alpha- and beta-chain diversity. The kit leverages SMART technology and employs a 5' RACE-like approach to capture complete V(D)J variable regions of TCR transcripts. Included in the kit are primers that incorporate Illumina-specific adaptor sequences during cDNA amplification. The protocol generates indexed libraries that are ready for sequencing on Illumina platforms.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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634431: SMARTer Human scTCR a/b Profiling Kit

634431: SMARTer Human scTCR a/b Profiling Kit

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Why single-cell TCR analysis is needed

Why single-cell TCR analysis is needed

Benefits of studying T cells at the single-cell level. Panel A. Schematic of a T-cell receptor comprised of an alpha chain (TCR-α) and a beta chain (TCR-β). Panel B. Schematic representing the difficulties of obtaining pairing information for alpha and beta chains from bulk sequencing data. Rare clonotypes can allow for assessing the pairing of some cells (clonotypes in orange). However, for the more highly expressed clonotypes (TCRA V2J1, TCRA V1J2, TCRB V3D1J1, TCRB V2D1J2) there are four possible pairing combinations.

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Bioanalyzer traces from example TCR libraries

Bioanalyzer traces from example TCR libraries

Electropherogram profiles of TCR sequencing libraries. Electropherograms from three different cell pools (see technical note for more details): Pool 9 (Panel A), 10 (Panel B), and 11 (Panel C). The variation in the profiles reflects the abundance of amplified TCRa (peak at ~900 bp) and TCRb (peak at ~700 bp). Panel D. Positive Control RNA TCR library (generated with 8 x 5 pg Control Jurkat Total RNA). Panel E. Negative Control TCR library shows no library is produced.

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Cell-type calling and alpha-beta pairing analysis of a mixed population of cells

Cell-type calling and alpha-beta pairing analysis of a mixed population of cells

Analysis of a mixed cell population from a 96-well plate. Panel A. Cell-type calling based on the identified clonotypes for each well. The seven omitted cells did not have clonotype calls for either TCRa or TCRb with read numbers that were above the threshold. Panel B. Analysis of pairing information. Paired TCR-αβ chains were obtained for 34 cells in the plate. Panel C. The alpha, beta, or alpha-beta pairing information represented as a percent of cells analyzed. Omitted cells were not included in this analysis.

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The SMARTer scTCR workflow uses optimized indexing to allow for pooling 96 cells into 12 libraries.

The SMARTer scTCR workflow uses optimized indexing to allow for pooling 96 cells into 12 libraries.

SMARTer Human scTCR a/b Profiling Kit workflow and pooling strategy. Panel A. First-strand cDNA synthesis is dT-primed (RT Primer) and performed by an MMLV-derived reverse transcriptase (RT), which adds nontemplated nucleotides to the 5' end of each mRNA template. The SMART-Seq Indexed Oligos anneal to these nontemplated nucleotides and serve as a template for the incorporation of an additional sequence of nucleotides into the first-strand cDNA by the RT (this is the template-switching step). Each of the eight different SMART-Seq Indexed Oligos provided in the kit each contains a unique six-base in-line index that serves as a cell barcode to allow downstream cell identification after pooling. The additional sequence added to the cDNA by the RT—referred to as the "SMART sequence"—serves as a primer-annealing site for subsequent rounds of PCR, ensuring that only sequences from full-length cDNAs undergo amplification. After pooling (described in Panel B) and a cleanup step, two rounds of gene-specific PCR are performed in succession to amplify cDNA sequences corresponding to variable regions of TCRa and/or TCRb transcripts. The first gene-specific PCR uses the amplified double-stranded cDNA as a template and includes a forward primer with complementarity to the SMART sequence—which also incorporates the Illumina Read 2 sequence (TCR Primer 1)—and reverse primers that are complementary to the constant (i.e., nonvariable) region of TCRa and TCRb (TCR a/b Human Primer 1). The second round of PCR takes the product from the first PCR as a template and uses a forward primer that binds to the Read 2 sequence added by the previous PCR step. The reverse primers bind in the constant region, internal to the PCR1 primers (TCR a/b Human Primer 2 Reverse HT Index) allowing amplification of the entire variable region and a portion of the constant region of TCRa and TCRb cDNA. The forward and reverse primers include adapter and index sequences that are compatible with the Illumina sequencing platform and allow for multiplexing of up to 96 samples in a single flow-cell lane. Panel B. Samples are pooled by column, such that each pool contains eight cells each with a differently indexed SMART-Seq Indexed Oligo. Different combinations of the Forward and Reverse HT indexes are used during PCR 2 to allow multiplexing of the samples into a single flow-cell lane (see the User Manual for more details).

640182 ICELL8® Human TCR a/b Profiling Reagent Kit Each USD $546.00

The ICELL8 Human TCR a/b Profiling Reagent Kit is required for the ICELL8 Human TCR a/b Profiling and ICELL8 cx Human TCR a/b Profiling workflows, which enables users to analyze T-cell receptor (TCR) diversity from single T cells using the ICELL8 platform. The kit leverages SMART technology and employs a 5' RACE-like approach to capture complete V(D)J variable regions of TCR transcripts. The reagents are dispensed into a ICELL8 TCR Chip (Cat. No. 640178) or ICELL8 cx TCR Chip (Cat. No. 640200) which includes 5,184 nanowells, containing 1,728 preprinted barcodes (each barcoded is printed three times on the chip) along with primers from the ICELL8 Human TCR a/b Profiling - Indexing Set (Cat. Nos. 640179, 640180 & 640181) that incorporate Illumina-specific adaptor sequences during cDNA amplification. The protocol generates indexed libraries that are ready for sequencing on Illumina platforms. The protocol also allows whole transcriptome library generation of 5' ends of all transcripts.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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640182: ICELL8 Human TCR a/b Profiling Reagent Kit

640182: ICELL8 Human TCR a/b Profiling Reagent Kit
635016 SMARTer® Human TCR a/b Profiling Kit 96 Rxns USD $6129.00

License Statement

ID Number  
275 SMART-Seq2 Technology. This product is sold under exclusive license from Ludwig Institute of Cancer Research, Ltd. and is covered by US Patent No. 10266894, Japanese Patent No. 6336080, and European Patent No. 3036336, and pending U.S. patent application and/or pending claims of foreign counterparts. For license information, please contact a Takara Bio USA, Inc. licensing representative by e-mail at licensing@takarabio.com.

The SMARTer Human TCR a/b Profiling Kit enables users to analyze T-cell receptor (TCR) diversity from input RNA samples. This kit is designed to work with a range of RNA input amounts depending on the sample type, and has been shown to generate high-quality libraries from as little as 10 ng to 3 μg of total RNA obtained from peripheral blood leukocytes, or from 50 to 10,000 purified T cells. As the name suggests, the kit can be used to generate data for both alpha- and beta-chain diversity, either in the same experiment or separately.

This kit leverages SMART technology and employs a 5' RACE-like approach to capture complete V(D)J variable regions of TCR transcripts. Included in the kit are primers that incorporate Illumina-specific adaptor sequences during cDNA amplification. The protocol generates indexed libraries that are ready for sequencing on Illumina platforms.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Sequencing reads on target and correlation of clonotype count data for varying sample input amounts

Sequencing reads on target and correlation of clonotype count data for varying sample input amounts
Sequencing reads on target and correlation of clonotype count data for varying sample input amounts. Panel A. Percentages of sequencing reads that map to CDR3 regions in either TCR-α (blue) or TCR-β (purple) or that represent off-target reads (gray). The experimental protocol was performed on three different amounts of peripheral blood RNA: 10 ng, 100 ng, and 1,000 ng. Panel B. Correlation of clonotype count data for 100 ng input RNA vs. 1,000 ng input RNA. Pearson (R) and Spearman (ρ) correlation coefficients are included.

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Assessing the sensitivity and reproducibility of the SMARTer approach

Assessing the sensitivity and reproducibility of the SMARTer approach
Assessing the sensitivity and reproducibility of the SMARTer approach. The experimental protocol was performed in replicate on PBMC RNA samples spiked at varying concentrations (10%, 1%, 0.1%, 0.01%, and 0.001%) with RNA obtained from a homogenous population of Leukemic Jurkat T cells (TRAV8-4-TRAJ3, TRBV12-3-TRBJ1-2 clonotype). Panel A. Correlation between concentration of spiked-in Jurkat RNA and number of TRBV12-3-TRBJ1-2-specific sequence reads. Numbers along the X-axis indicate serial-diluted concentrations of spiked-in Jurkat RNA (by mass): 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = 0.01%; 5 = 0.0.01. Count data for TRBV12-3-TRBJ1-2-specific sequence reads were normalized by subtracting the number of corresponding reads obtained for negative control samples consisting of unspiked PBMC RNA. Normalized count data were then Log<sub>10</sub> transformed. Circles and triangles correspond to experimental replicates for each sample concentration. Results of linear regression analysis are indicated in the upper right region of the graph. Panel B. Count data, signal-to-noise ratios, and statistical analysis for TRBV12-3-TRBJ1-2-specific sequence reads obtained from spiked RNA samples. Signal-to-noise ratios were generated using the mean counts of TRBV12-3-TRBJ1-2-specific sequence reads for each pair of experimental replicates. Rows highlighted in yellow include concentrations of spiked-in Jurkat RNA for which statistically significant elevations in TRBV12-3-TRBJ1-2-specific sequence reads were detected relative to background counts observed for unspiked negative control RNA samples.

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Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach

Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach
Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach. Panel A. Reverse transcription and PCR amplification of TCR subunit mRNA sequences. First-strand cDNA synthesis is primed by the TCR dT Primer and performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5′ end of each mRNA molecule, the RT adds non-templated nucleotides to the first-strand cDNA. The SMART-Seq v4 Oligonucleotide contains a sequence that is complementary to the non-templated nucleotides added by the RT, and hybridizes to the first-strand cDNA. In the template-switching step, the RT uses the remainder of the SMART-Seq v4 Oligonucleotide as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. Full-length variable regions of TCR cDNA are selectively amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (SMART Primer 1) and the constant region(s) of TCR-α and/or TCR-β subunits (TCR a/b Human Primer 1). A subsequent round of PCR is performed to further amplify variable regions of TCR-α and/or TCR-β subunits and incorporate adapter sequences, using TCR Primer 2 and TCR a/b Human Primer 2. Included in the primers are adapter and index sequences (read 2 + i7 + P7 and read 1 + i5 + P5, respectively) that are compatible with the Illumina sequencing platform. Following purification, size selection, and quality analysis, the TCR cDNA library is ready for sequencing. Panel B. Semi-nested PCR approach for amplification of TCR-α and/or TCR-β subunits. The primer pairs used for the first round of amplification capture the entire variable region(s) and most of the constant region(s) of TCR-α and/or TCR-β cDNA. The second round of amplification retains the entire variable region(s) of TCR-α and/or TCR-β cDNA, and a smaller portion of the constant region(s). The anticipated size of final TCR library cDNA (inserts + adapters) is ~700–800bp.


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