Single-cell sequencing of antigen-specific B cells

Six days after adoptive transfer, individual transduced SW_HEL B cells were recovered from the spleens of host mice into 96-well PCR plates (Bio-Rad) as GFP^+ve cells that bound HEL conjugated to Alexa Fluor 647 (30 (link)). Following cell lysis and protein digestion, the VDJ_H-region of the single SW_HEL allele present in each well was amplified by nested PCR, as described (30 (link)). PCR products from ≤60 wells per host in which amplification was successful were Sanger sequenced by Macrogen (South Korea). Mutations where secondary peaks formed <30% of the signal were confirmed using Sequencher software (version 5.1, Gene Codes Corporation). Processed sequences were sorted into phylogenetic trees (using neighbor joining and uncorrected ‘p’) with MacVector software (version 12.7.5 MacVector Inc) to check for clonal dynasties, then collated for a window spanning nucleotides 17–539 (counting from the translation start ATG codon as bases 1–3) using custom Microsoft Excel spreadsheets (version 15.23 for Mac, Microsoft Corporation). Statistical analyses of mutation data exported from Excel were performed using Prism software (version 7.0a for Mac, GraphPad Software).