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Introduction Single-cell T-cell receptor (scTCR) clonotype analysis permits the determination of the specific TCR alpha-beta (α/β) chain pairing expressed on each cell. This pairing information allows researchers to gain insight into Tcell heterogeneity and plasticity, determine the contribution of the pairing to antigen specificity of the individual TCR, and design therapeutic antibodies. Here we employ a 5’-RACE-like approach and SMART technology, in conjunction with two novel next-generation sequencing (NGS) library preparation kits, using the same primer pairs, to capture full-length variable regions of TCR-α and -β chains. Material and methods Method 1, using the SMARTer Human scTCRa/b Profiling Kit, permits NGS library preparation of FACS-sorted cells in 96-well plates. Method 2 is an adaptation of the process above that scales to ~1200 single cells using the SMARTer ICELL8 Single-Cell System, which enables single-cell isolation and nanoliter PCR in a nanowell chip. Results and discussions We present data showing α/β pairing from Jurkat, CCRF, PBMCs, and CD4 +T cells. In addition to the sensitivity of this method, the ability to pool the cDNA from 96wells into 12 sequencing libraries adds to the ease of use. Consistent with immunology reports, unstimulated CCRF-CEM cells examined with this kit expressed a TCR-β but not a TCR-α chain. scTCR pofiling with SMARTer ICELL8 Single-Cell System as a proof of principle of scTCRα/β was performed on Jurkat cells and CCRF-CEM cells. Jurkat cells and CCRF-CEM cells were processed using an ICELL8 chip preprinted with barcoded oligos. Paired TCRα/β Jurkat clonotypes were detected in 77% and 87% cells in mixed and single cell populations, respectively. Conclusion The ability of the core biochemistry and PCR components of these kits to be used with either FACS-sorted cells in 96-well plates or >1,000 cells in novel ICELL8 chips (in development) points to the general utility and scalability of this approach in understanding paired scTCR clonotype diversity. |
