DNA sequencing for multiple rounds of the pMHC-yeast display selection for 2W, Yae and Fat TCR

A peptide library was displayed on Ab MHC class II allele expressed on yeast. TCRs of interest were used to select and enrich for cognate peptide-MHC. A naive pMHC-yeast display library was selected with biotinylated TCRs of interest present on magnetic beads (Miltenyi) or fluorescently labelled streptavidin. After 4 rounds of TCR selection, DNA was extracted using zymolase for all rounds of selected yeast, enriched for cognate pMHC, as well as the naive library. Isolated DNA/plasmid was amplified by PCR with appropriate barcodes and sequencing was performed using a Miseq.