BCR sequencig results from hD3-3/JH6 mouse immunization with Env gp151 MPER mRNA

Homozygous hD3-3/JH6 mice were injected with 10 µg mRNA LNPs encoding Env gp151 MPER I.M. After six weeks, mice were euthanized and splenoctyes and lymphocytes were collected for B cell sorting by FACS. Cells were bulk sorted and processed for BCR sequencing via the 10x Genomics Single Cell Immune Profiling according to kit protocols. Pooled libraries were sequenced on an Illumina NextSeq 2000 using a 100-cycle P3 reagent kit. Raw sequencing data were demultiplexed, processed into assembled VDJ contigs and counts matrix files, and assigned to specific animal IDs based on TotalSeq-C antibody hashtag counts. 

Gene assignment, annotation, and formatting into Adaptive Immune Receptor Repertoire (AIRR) format for paired heavy and light chain antibody sequences was performed using Sequencing Analysis and Data library for Immunoinformatics Exploration (SADIE) with a custom germline reference database that included all known mouse antibody genes plus the human IGHD3-3 and IGHJ6 genes that were knocked into hD3-3/JH6 mice. Eight antibodies from each animal were randomly selected for in vitro analysis, except for one animal from which only two total heavy/light pairs were recovered. In addition, 12 antibodies with long HCDR3s (>19 aa) were selected for in vitro characterization.