Shifted gate sample from EB81.103 TCR-T + WES (non-CRISPR edited K562 chassis) round 1 Tope-seq experiment
Cells sorted from matched Shifted and Unshifted gates in each replicate Tope-seq screening experiment were processed for gDNA isolation using DNAzol reagent according to manufacturers protocol. To prepare samples for sequencing, two rounds of PCR were conducted. First round PCR was run for 25 cycles with Phusion polymerase (NEB) and Illumina adapter primers, F: 5-ACGACGCTCTTCCGATCT-3 and R: 5-CGTGTGCTCTTCCGATCT-3. For second round PCR, 100 ng of column-cleaned first round product was used as template and amplified for 5 cycles using primers from NEBNext Multiplex Oligo Set, uniquely barcoding each gate sample of each replicate experiment.