GSM6276325: CDRH3_IGHV1-69_L54; Mus musculus; OTHER
Mouse spleens were removed and single cell suspensions were obtained using 70um cell strainer. CDRH3 diversity within the transgenic mice was measured by deep sequencing the B cell receptors (BCRs) from antigen naïve IgM B cells in bulk according to previous methods (Sangesland et al., 2019; Trombetta et al., 2014). Briefly, BCR libraries were enriched from whole transcriptome amplification (WTA) products from B cells using the Smart-Seq2 protocol. Following WTA, antibody heavy chains were PCR amplified using a forward primer specific for the FR3 region within the VH gene of interest (IGHV1-69) and reverse primers against the heavy constant regions. These primers were attached to the Illumina P7 (FR3 region) and P5 (constant region). Heavy chain sequences were further amplified using step-out PCR with the Illumina sequencing adapters. The resulting PCR products (FR3 through CDRH3) were sequenced using the Illumina Miseq system (MiSeq Reagent Kit v2 500-cycle; Illumina, San Diego, US).