Abstract Identification of correlated antigens recognized by T cell and its T cell receptor (TCR) sequences at single cell level is extremely useful to the understanding and treating immune-related diseases. Although CyTOF has pushed the number of antigenic peptides that can be interrogated at once to around 100, the destructive nature of CyTOF prohibits linking pMHCs recognized by a T cell with its TCR receptor sequences. Here we describe a new technology, TetATCR-Seq for Tetramer Associated TCR sequencing, that is capable of de novo generating a large amount of peptides and mapping antigen recognition with single cell TCR sequences by DNA barcoded tetramers. It evolves three major steps: 1) de novo peptide generation, 2) DNA barcoded tetramer generation, and 3) single cell sequencing of tetramer DNA barcode and TCR sequences. Using a published set of cancer neo-antigen sequences, we showed that these peptides and their wildtype counterparts can be quickly generated. Resulted tetramers can be used to clearly identify a population of neo-antigen only or wild type antigen only or double positive T cells. Subsequent single cell TCR sequencing showed that TCR paired a and b chain sequences can be obtained in more than 80% of single T cells. Simultaneous tetramer DNA barcode sequencing showed that many T cells are found to cross-reactive to both neo-antigen and its wild type counterpart, suggesting the necessity of strict screening for neo-antigen only TCR in adoptive cell transfer therapy. In summary, TetaTCR-seq enables one to quickly survey antigen repertoire again TCR repertoire at single cell level for a large number of peptides. It is a useful technology to screen for T cell cross-reactivity and select neo-antigen specific TCR for cancer immunotherapy.