T cells play a vital role in the adaptive immune system's ability to combat invading pathogens and establish defenses against various diseases. T-cell receptors (TCRs) form the basis of specificity for each individual T cell. To advance discovery in pathogenesis, host response, and potential therapies for cancer, infectious diseases, and autoimmune disorders, we need powerful tools that can decode TCR sequences from both TCR alpha and beta chains in mouse samples.
Our new SMART-Seq Mouse TCR (with UMIs), referred to as the “mTCRv2” kit, is designed to address this need. We have improved the chemistry to ensure the new kit has:
- Unparalleled sensitivity—for rare TCR clonotype calling
- Impeccable reliability—with UMI-based error correction
- Simple methodology—with a streamlined protocol for pooling
Applications of mTCRv2 include but are not limited to researching:
- The development of lymphocytes—how different factors drive compartmentalization, differentiation, and distribution of T cells in different mouse tissues
- Biomarkers in mouse disease models—how TCR signatures function as biomarkers for prognosis, diagnosis, and treatment of autoimmune diseases like Type 1 diabetes and rheumatoid arthritis
- Responses to cancer immunotherapy—how relative abundance of different T-cell clones affects sensitivity to cancer immunotherapy
- Host response in infectious diseases—how T cells influence the responsiveness to antiretroviral therapy or cause remission in certain cases
A SMART-based approach for sensitive and unbiased detection of TCR transcripts
Exactly how does mTCRv2 technology work? First-strand cDNA synthesis is dT-primed and results in the addition of non-templated nucleotides. The TCR SMART UMI Oligo, which has UMI sequences incorporated, then anneals to the non-templated oligonucleotides added by the reverse transcriptase. These oligonucleotides serve as a new template for primers to bind during PCR1 to amplify the entire variable region and considerable portion of the constant region from both TCR alpha and beta chains (Figure 1, Panel A). Products from PCR1 are used as templates for the second round of semi-nested PCR, in which the entire variable region gets amplified and indexed with the UDI sets. Following bead purification, size selection, and quality check, the library is ready for Illumina sequencing on various Illumina sequencers (Figure 1, Panel B).
Figure 1. SMART-Seq Mouse TCR (with UMIs) workflow. Panel A. First-strand cDNA synthesis is dT-primed and performed by the MMLV-derived SMARTScribe Reverse Transcriptase (RT), which adds non-templated nucleotides (XXXXX) upon reaching the 5’ end of each mRNA template. The TCR SMART UMI Oligo anneals to these nucleotides, allowing it to serve as a template for the incorporation of the SMART adapter sequences (light green) and UMI (yellow) into the first-strand cDNA. After this template switching step is completed, the first round of PCR (PCR1) is carried out with the forward primer (mTCR PCR1 Universal Forward) adding the Read 2 sequence (dark green), while the reverse primer (mTCRa/mTCRb PCR1 reverse, orange) anneals within the constant region. PCR1 ensures amplification of all TRA/TRB transcripts in an unbiased manner. PCR2 further enriches TRA/TRB transcripts, adds UDIs, and decreases final amplicon sizes for optimal clustering on Illumina sequencers. The UDIs include adapter and index sequences compatible with various Illumina sequencers and allow for multiplexing of up to 384 TCR libraries in a single flow cell lane.
