Amplification and Sequencing of scFv/scTGP

The plasmid DNA collected from the yeast cells of the anti-CDK2A and anti-USP11 second sort outputs obtained both as scFvs and as scTGPs were used as templates for PCR amplifications targeting the HCDR3 region of the scFvs and scTGPs. The HCDR3s were amplified with a set of forward primers carrying one of the Ion Torrent sequencing adaptors and mapping to the framework region upstream of HCDR3 in combination with a barcoded reverse primer mapping to the SV5 tag region of the yeast display vector and carrying the second adaptor necessary for sequencing. Primer sequences and detailed method are described by D’Angelo et al [37 (link)]. After amplification with the proofreading Phusion polymerase (NEB), gel extraction, and quantification (Q-bit, HS-DNA kit, Invitrogen), the sequencing libraries were processed using the Ion Xpress Amplicon library protocol and then prepared for sequencing on the Ion 316 Chip (Life Technologies). The analysis of the sequences was performed using the AbMining Toolbox as described by D’Angelo et al [37 (link)].