The VH and VL coding genes from each round of a given experiment were amplified from pooled phagemid preparations. For PCR, the following primers with Illumina adapters were used: 5′leadVH—TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTGCCCAACCAGCCATGGCC; 3′VH_rev—GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGATGGGCCCTTGGTGGAGGC; 5′Vkappa—TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGCCCAGGCGGCCGAGCTC; and 3′Vkappa_rev—GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGACAGATGGTGCAGCCACAGT.
The reactions were performed using Platinum Taq DNA Polymerase (Invitrogen) according to the manufacturer’s instructions and the cycling was as follows: 95°C for 2 minutes; 30 cycles of 95°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute, followed by an extra 5-minute incubation at 72°C. The amplicons were analyzed in 0.8% agarose gel from where it was extracted and purified using UltrafreeDA columns (Millipore), according to the manufacturer’s instructions prior to NGS sequencing.
The reactions were performed using Platinum Taq DNA Polymerase (Invitrogen) according to the manufacturer’s instructions and the cycling was as follows: 95°C for 2 minutes; 30 cycles of 95°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute, followed by an extra 5-minute incubation at 72°C. The amplicons were analyzed in 0.8% agarose gel from where it was extracted and purified using UltrafreeDA columns (Millipore), according to the manufacturer’s instructions prior to NGS sequencing.