USR_template_replicates

Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies, thus it is essential to establish the reliability of antibody NGS data. Therefore, two conditions of immunological relevance were assessed for NGS reproducibility. First, we pooled spleen plasmablasts and plasma cells (CDR138-enriched) and bone marrow plasma cells (CD45R-depleted and CD138-enriched 4) of 1 mouse (1M) immunized with NP-CGG (chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl) and sacrificed 14 days post-immunization (dpi). The thus created cell pool contained approximately 3 x 106 viable ASCs. Second, in order to model extreme antibody diversity, we repeated the same cell isolation procedure from 9 immunized mice (9M) resulting in an ASC pool of approximately 2.5 x 10^7 viable cells. From isolated cells, we recovered total RNA and used RT-PCR to amplify expressed rearranged IgG variable heavy VDJ genes; PCR was performed using a well-characterized and utilized primer set based on variable framework 1 region forward primers and one IgG constant region 1 reverse primer (covering all IgG isotypes) 43. Similarly to previously published methods (Vollmers et al., 2013, PNAS), Illumina adaptors were added during the PCR step by using a direct addition approach where adaptors are added at the 5’ end of the gene-specific portion of the primer set, thus circumventing the need for ligation of adaptors following PCR. For each of the two conditions (1M/9M), triplicates were prepared, where a triplicate signifies three separately indexed samples prepared from the same starting cDNA pool; thus variable region PCR was independently performed in each of the triplicates. All six samples (triplicates of 1M and 9M) were sequenced using the Illumina MiSeq platform with 250bp paired-end reads.