FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells
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 sequ...
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Frontiers Media S.A.
2024-12-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1505971/full |
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| author | Sakina Ado Chuang Dong Noudjoud Attaf Myriam Moussa Agathe Carrier Agathe Carrier Pierre Milpied Jean-Marc Navarro |
| author_facet | Sakina Ado Chuang Dong Noudjoud Attaf Myriam Moussa Agathe Carrier Agathe Carrier Pierre Milpied Jean-Marc Navarro |
| author_sort | Sakina Ado |
| collection | DOAJ |
| description | 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 96-well 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, cost-effective, 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. |
| format | Article |
| id | doaj-art-a70950f1c8fe4a6695e018c76cec7f77 |
| institution | OA Journals |
| issn | 1664-3224 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Immunology |
| spelling | doaj-art-a70950f1c8fe4a6695e018c76cec7f772025-08-20T02:36:38ZengFrontiers Media S.A.Frontiers in Immunology1664-32242024-12-011510.3389/fimmu.2024.15059711505971FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cellsSakina Ado0Chuang Dong1Noudjoud Attaf2Myriam Moussa3Agathe Carrier4Agathe Carrier5Pierre Milpied6Jean-Marc Navarro7Aix Marseille Université, CNRS, INSERM, Centre d’Immunologie de Marseille-Luminy, Marseille, FranceAix Marseille Université, CNRS, INSERM, Centre d’Immunologie de Marseille-Luminy, Marseille, FranceAix Marseille Université, CNRS, INSERM, Centre d’Immunologie de Marseille-Luminy, Marseille, FranceAix Marseille Université, CNRS, INSERM, Centre d’Immunologie de Marseille-Luminy, Marseille, FranceAix Marseille Université, CNRS, INSERM, Centre d’Immunologie de Marseille-Luminy, Marseille, FranceParis-Saclay University, Inserm, Gustave Roussy, Tumour Immunology and Anti-Cancer Immunotherapy, Villejuif, FranceAix Marseille Université, CNRS, INSERM, Centre d’Immunologie de Marseille-Luminy, Marseille, FranceAix Marseille Université, CNRS, INSERM, Centre d’Immunologie de Marseille-Luminy, Marseille, FranceParallel 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 96-well 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, cost-effective, 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.https://www.frontiersin.org/articles/10.3389/fimmu.2024.1505971/fullB cellssingle-cell RNA-seqantibody cloningBCR sequencingantigen specificity |
| spellingShingle | Sakina Ado Chuang Dong Noudjoud Attaf Myriam Moussa Agathe Carrier Agathe Carrier Pierre Milpied Jean-Marc Navarro FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells Frontiers in Immunology B cells single-cell RNA-seq antibody cloning BCR sequencing antigen specificity |
| title | FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells |
| title_full | FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells |
| title_fullStr | FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells |
| title_full_unstemmed | FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells |
| title_short | FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells |
| title_sort | fb5p seq mabs monoclonal antibody production from fb5p seq libraries for integrative single cell analysis of b cells |
| topic | B cells single-cell RNA-seq antibody cloning BCR sequencing antigen specificity |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1505971/full |
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