B cells are an essential part of the adaptive immune response, functioning via B-cell receptors (BCRs) expressed on their surface. Each B cell expresses a different BCR that allows it to recognize molecular patterns in pathogens. Development of BCRs (Figure 1) is a multistep process in which the progenitor cell undergoes V(D)J recombination in the germline and additional somatic hypermutations (SHM), resulting in a final product with a specific CDR3 (complementarity determining region 3) sequence in the hypervariable region of the immunoglobulin. The unique CDR3 sequence in BCRs is critical for dictating antigen specificity. Taken together, the molecular events described above facilitate receptor diversity and the generation of heavy (H) chain isotypes. BCR diversity enables B cells to recognize and respond to a wide range of pathogens. Upon exposure to a stimulus or stimuli, the lambda (L) and kappa (K) light chain genes of the BCR undergo rearrangements to generate specific gene segments. This further development results in different light chain isotypes being generated from the same B-cell clone.
Recently, next generation sequencing (NGS) approaches for profiling B-cell repertoires have provided valuable insights into the adaptive immune response and antibody engineering. There are two major approaches used in profiling B-cell repertoires—multiplex PCR and 5' RACE combined with NGS. While multiplexing allows you to amplify multiple BCR genes in one reaction, it may prove challenging with regard to sensitivity, specificity, and biases in amplification of certain sequences, all of which can lead to difficulties in accurate identification of isotypes. On the other hand, the 5'-RACE method reduces variability and allows for priming from the constant region of BCR heavy or light chains. However, the burden of designing optimized primers would still be left to the individual user in this case. The new SMARTer Mouse BCR IgG H/K/L Profiling Kit (SMARTer mouse BCR kit) solves this problem by combining the benefits of 5' RACE with gene-specific amplification (Figure 2) to provide a highly sensitive and reproducible method for profiling B-cell repertoires, by capturing complete V(D)J variable regions of BCR transcripts. The high sensitivity of the kit allows for accurate identification of top clonotypes and reliable assignment of isotype in a majority of cases, based on the sequencing of the H, K, and L chains.
Features of the kit:
- Starts with 10 ng–3 µg of total RNA from spleen, lymph node, PBMCs, and hybridomas
- 5' RACE-based approach combined with gene-specific primer amplification of cDNA libraries is optimized for sensitive and specific clonotype detection
- Optimized library generation workflow for amplification of light chains for mapping and identification of clonotypes
- Accurate amplification of mouse IgG subclasses and identification via sequencing in a majority of cases
The SMARTer Mouse BCR IgG H/K/L Profiling Kit leverages SMART technology (Switching Mechanism at 5' End of RNA Template) and employs a 5' RACE-like approach to capture complete V(D)J variable regions of BCR transcripts. First-strand cDNA synthesis is dT-primed, and the template-switching activity ensures that only sequences from full-length cDNAs undergo PCR amplification (Figure 2). Two rounds of PCR are then performed in succession to amplify cDNA sequences corresponding to the variable regions of BCR IgG heavy chain or BCR light chain (kappa or lambda) transcripts (Figure 2, Panel B). The first PCR uses the first-strand cDNA as a template, and heavy or light chains are amplified in separate reactions. This PCR specifically amplifies the entire variable region and a portion of the constant region of BCR heavy or light chain cDNA. The second PCR uses the first PCR product as a template and uses semi-nested primers to amplify the entire variable region and a portion of the constant region of heavy or light chain cDNA. Following post-PCR purification, size selection, and quality analysis, the library is ready for sequencing on Illumina platforms.