The Experimental Procedure of Somatic Hypermutation Analysis in Splenic B Cells During Primary Response

Somatic hypermutation (SHM) is a critical process in adaptive immunity, enabling the generation of high-affinity antibodies through targeted mutations in immunoglobulin variable (IgV) regions. Here, we provide a comprehensive workflow combining immunization, molecular biology, and bioinformatics to investigate SHM mechanisms and outcomes. This is a general protocol for studying SHM at the VH186.2 region in C57BL/6 mice. While this method can be applied broadly, this chapter will detail the protocol used to test the effects of exonuclease 1(EXO1) knock-in mutation (Exo1^D173A, or Exo1^DA) or knock-out (KO) on hypermutation post-immunization by immunizing age-matched mice with NP(33)-CGG on alum. We start by immunizing and sacrificing the animals to obtain spleens for RNA extraction. We then create cDNA libraries and investigate VH186.2 region mutation to analyze SHM.