S1). The PCR products for each variable region were pooled according to the natural distribution as described on V-Base. The light chain variable regions were cloned first using restriction digest with SfiI and AvrII for Vλ and SfiI and BsiWI for Vκ and transformed into electrocompetent TG1 cells (48 μg DNA in 48 200 μL Apoptosis Compound Library cost transformations for Vκ and 65 μg DNA in 65 200 μL transformations for Vλ). Transformations were spread on 2xYT medium with 2% glucose and 100 μg/mL carbenicillin, which were incubated overnight at 30 °C. The following morning the bacteria were scraped from the plates, combined and plasmid DNA purified

with the GenElute™ HP Maxiprep Kit (Sigma-Aldrich). The resulting DNA was prepared for cloning VH with NcoI-HF and NheI-HF. The ligated DNA was cleaned with the Wizard® SV Gel and PCR Clean-up system (Promega) and transformed into electrocompetent TG1 cells (66 μg DNA in 66 200 μL transformations for Vκ and 100 μg DNA in 100 200 μL transformations for Vλ). Transformations were spread on 2xYT medium PD98059 manufacturer with 2% glucose and 100 μg/mL carbenicillin, which were incubated overnight at 30 °C. The following morning the bacteria were scraped from the plates, combined, and stored in 15% glycerol 2xYT at − 80 °C. The scFv library was constructed similarly to the above described Fab library with the following changes. Primer sequences are listed in Table S3 and Table S4. cDNA from 20 PBMC samples, 8 bone marrow

samples, 1 lymph node sample, and 1 spleen sample ifenprodil were used. The reverse secondary PCR primers for VH and forward secondary primers for Vκ and Vλ had complementary extensions for an AST(G4S)3 linker and the forward secondary PCR

primers for VH and reverse secondary primers for Vκ and Vλ had sequences to add flanking SfiI restriction sites. A tertiary PCR step was then done to assemble the full length scFv fragment, which was next cloned into pXHMV-scFv ( Fig. S1) using the SfiI sites. The ligated DNA was transformed into electrocompetent TG1 cells (147 μg DNA in 120 200 μL transformations for Vκ and 44 μg DNA in 40 200 μL transformations for Vλ). Transformations were spread on 2xYT medium with 2% glucose and 100 μg/mL carbenicillin, which were incubated overnight at 30 °C. The following morning the bacteria were scraped from the plates, combined, and stored in 15% glycerol 2xYT at − 80 °C. Both XFab1 and XscFv2 phage libraries were rescued using a modification of the standard protocol (Marks et al., 1991). XFab1 was rescued in four batches (two for XFab1λ and two for XFab1κ) each starting with 5-fold more bacteria than the sub-library size. XscFv2 was rescued in five batches (two for XscFv2λ and three for XscFv2κ) with XscFv2λ starting 5-fold more bacteria than the sub-library size and XscFv2κ starting with 3.33-fold more bacteria than the sub-library size. For all rescue batches, cultures were seeded at a starting density of 0.