Sakina Ado Chuang Dong Noudjoud Attaf Myriam Moussa Agathe Carrier Pierre Milpied ^* Jean-Marc Navarro ^*

• INSERM U1104 Centre d'immunologie de Marseille-Luminy (CIML), Marseille, Provence-Alpes-Côte d'Azur, France

Parallel analysis of phenotype, transcriptome and antigen receptor sequence in single B cells is a useful method for tracking B cell activation and maturation during immune responses. However, in most cases, the specificity and affinity of the B cell antigen receptor cannot be inferred from its sequence. Antibody cloning and expression from single B cells is then required for functional assays. Here we propose a method that integrates FACS-based 5'-end single-cell RNA sequencing (FB5P-seq) and monoclonal antibody cloning for integrative analysis of single B cells. Starting from a cell suspension, single B cells are FACS-sorted into 96well plates for reverse transcription, cDNA barcoding and amplification. A fraction of the single-cell cDNA is used for preparing 5'-end RNA-seq libraries that are sequenced for retrieving transcriptome-wide gene expression and paired BCR sequences. The archived cDNA of selected cells of interest is used as input for cloning heavy and light chain variable regions into antibody expression plasmid vectors. The corresponding monoclonal antibodies are produced by transient transfection of a eukaryotic producing cell line and purified for functional assays. We provide detailed step-by-step instructions and describe results obtained on ovalbumin-specific murine germinal center B cells after immunization. Our method is robust, flexible, costeffective, and applicable to different B cell types and species. We anticipate it will be useful for mapping antigen specificity and affinity of rare B cell subsets characterized by defined gene expression and/or antigen receptor sequence.