Dynabead myone streptavidin t1 conjugated magnetic beads

Patient serum was incubated with the fully random 12-mer bacterial display peptide library (1 × 10¹⁰ diversity, 10-fold oversampled) at a 1:25 dilution in a 96-well deep-well plate format. Antibody-bound bacterial clones were selected with 50 µL Protein A/G Sera-Mag SpeedBeads (GE Life Sciences) or by incubation with a biotinylated anti-human IgM antibody (Jackson ImmunoResearch; 1:100 dilution) followed by a second incubation with 100 μL Dynabead MyOne Streptavidin T1 conjugated magnetic beads (ThermoFisher; 65602). The selected bacterial pools were resuspended in growth media and incubated at 37 °C with shaking overnight at 300 rpm to propagate the bacteria. Plasmid purification, PCR amplification of peptide encoding DNA, and barcoding with Illumina well-specific PCR indices were performed as previously described (49 (link)). Samples were normalized to a final concentration of 4 nM, pooled (94 samples per sequencing run), and sequenced on the Illumina NextSeq500. A detailed description of the SERA technology is described elsewhere (49 (link)).

A detailed description of the SERA assay has been published^{26 (link)}. For this study, serum or plasma was incubated with a fully random 12-mer bacterial display peptide library (1 × 10¹⁰ diversity, 10-fold oversampled) at a 1:25 dilution in a 96-well, deep well plate format. Antibody-bound bacterial clones were selected with 50 µL Protein A/G Sera-Mag SpeedBeads (GE Life Sciences, cat#17152104010350) (IgG) or by incubation with a biotinylated anti-human IgM antibody (Jackson ImmunoResearch, cat# 709-066-073) final assay dilution 1:100, followed by a second incubation with 50 µl Dynabead MyOne Streptavidin T1 conjugated magnetic beads (IgM) (Thermo-Fisher 65602). The selected bacterial pools were resuspended in growth media and incubated at 37 °C shaking overnight at 300 RPM to propagate the bacteria. Plasmid purification, PCR amplification of peptide-encoding DNA, barcoding with well-specific indices was performed as described^{26 (link)}. Samples were normalized to a final concentration of 4 nM for each pool and run on the Illumina NextSeq500. Every 96-well plate of samples processed for this study contained healthy control run standards to assess and evaluate assay reproducibility and possible batch effects.