In both pairing methods (single cell RT-PCR and pairSEQ) cDNA is used as template for multiplex PCR using TCRA and TCRB gene-specific primers. The resulting PCR product contains the 3’ end of the variable region and the full CDR3 region of matching TCRA and TCRB genes. These partial TCR sequences were analyzed with IMGT/V-Quest tool (http://www.imgt.org/IMGT) which identified the TRAV and TRBV families with the highest likelihood to contain the segment found with our pairing methods. Utilizing the IMGT database we reconstructed the full length TRAV and TRBV regions for each pairs. In regards to the constant regions we used modified murine TRAC and TRBC sequences to improve stability and avoid mismatches with the endogenous human TCR after transduction into human T-cells (22 (link)). Full TCR genes were synthetized and a 2A peptide (23 (link)) was introduced between the TCRB and TCRA chain to ensure a comparable expression efficiency of the 2 chains. The resulting TCRB-TCRA gene blocks were cloned into either a gamma-retroviral expression vector (24 (link),25 (link)) or for the following TCR pairs: 2650-1, 2650-3, 2650-4, 2650-5, 2650-6, 2650-7, 2650-9, 3903-3A1, 3903-3A2, 3992-1, 3992-2, 3992-3, 3992-4, 3992-5 and 3998-1 into a non-viral Sleeping Beauty transposon system (26 (link),27 ). The expression of the TCRB was evaluated with an anti-murine TCRB Ab.