Phage Library Immunoprecipitation and Sequencing

The T7-HCoV-56-mer phage library was generously provided to us by Dr. Stephen J. Elledge at Harvard Medical School, and it was previously described [29 (link)]. Phage immunoprecipitation and sequencing (PhIP-Seq) have been previously described by us and others [26 (link),27 (link),29 (link),50 (link)]. Briefly, the library was amplified and titered per the manufacturer’s instructions (Novagen T7Select System, Burlington, MA, USA). Then, 1.4 × 10⁹ pfu of the library (≈2 × 10⁵ pfu/library member) was mixed with plasma containing 2 µg of total IgG. The phage–antibody complexes were immunoprecipitated using Protein A and Protein G magnetic beads (10008D/9D, Invitrogen, Boston, MA, USA) and a magnetic separation rack (S1511S, NEB, Ipswich, MA, USA). After immunoprecipitation, the beads were resuspended in 40 µL of nuclease-free water, heated to 95 °C to lyse the phage, and the phage DNA was PCR amplified. During PCR amplification, each well of the 96-well plate was barcoded, and Illumina adaptors were added. The 376bp amplicon was then gel-extracted and sent to the UNMC Genomics core for quality check and sequencing. Finally, the clustering and sequencing were performed on an Illumina NextSeq550 using the 50-cycle, single-end protocol (Mid-output flow-cell). The run was monitored by Illumina Sequence Analysis Viewer, and the final FASTQ files were generated after de-multiplexing.