GSM4668360: BCR39_Bulk_IGHV1-2; Mus musculus; RNA-Seq
Mouse spleens were removed and single cell suspensions were obtained using 70 um 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 our previously deployed method (Sangesland et al., 2019; Trombetta et al., 2014). Briefly, BCR libraries were enriched from B cell whole transcriptome amplification (WTA) products using the Smart-Seq2 protocol. Following WTA, antibody heavy chains were PCR amplified (FR3 to CDR3) using a forward primer specific for the FR3 region within the VH gene of interest (IGHV1-69 or IGHV1-2) and reverse primers against the heavy constant regions. These primers were attached to the Illumina P7 (FR3 region) and P5 (constant region) as described previously (Sangesland et al., 2019). We then further amplified the heavy chain sequences 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).
GEO Accession
GSM4668360