Scholarlink

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.

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

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

  • 实验方案

  • 发表日期：

    2025年

查看全文
  • Sandy Al Hayek,
  • Antoine Praité,
  • Jean-Marie Lambert,
  • Sébastien Bender,
  • Laurent Delpy

摘要

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.