5'RACE Method for RNA-Based Sequencing of the Mouse Immunoglobulin Repertoire.

This chapter outlines a detailed methodology for high-throughput sequencing of the immunoglobulin (Ig) repertoire from mouse RNA samples. We describe RNA extraction from splenic B cells, followed by the conversion into cDNA using the 5'RACE (Rapid Amplification of cDNA Ends) technique and PCR amplification of the Ig heavy and light chain repertoire. The amplified products are prepared using specific primers for Illumina next-generation sequencing (NGS) and unique molecular identifiers (UMI) as molecular barcodes. The protocol includes stringent quality control steps to ensure the integrity of RNA and the accuracy of amplification. We describe the amplification of Ighm and all Ighg subclasses, for Ig heavy chains and for the predominant Igκ light chain isotype. RNA-based 5'RACE-RepSeq proves very useful to analyze the diversity and clonality of Ig repertoire, as well as somatic hypermutations, providing insights into the adaptive immune response. This method enables the high-throughput characterization of the mouse Ig repertoire in immunological contexts.