A streamlined approach for identifying and reconstructing TCRs, and testing for specificity to candidate antigens. T cells reactive against antigens are isolated by IFNγ secretion and submitted for single-cell paired TCRαβ sequencing. (A) Dominant TCR clonotypes are individually cloned by joining variable chain plasmid library components and an on-demand oligonucleotide encoding the CDR3 regions (supplemental Figure 1B-D). Linker sequence includes furin, SGSG, and F2A sequence. (B) TCRs are expressed in Jurkat∆αβ cells with an NFAT-luciferase construct (Jurkat∆αβ reporter cells; supplemental Figure 1A,E). (C) TCRs are screened for specificity against candidate antigens, with detection based on IL-2 secretion, CD69 expression, or luciferase activity.