FB5P-seq – FACS-based 5-prime end single-cell RNA-Seq

Single-cell RNA sequencing (scRNA-seq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on B and T cells of the adaptive immune system, scRNA-seq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (BCR or TCR, respectively) and link it to the functional state inferred from transcriptome analysis.

Researchers at the Centre d’Immunologie de Marseille-Luminy introduce FB5P-seq, a FACS-based 5′-end scRNA-seq method for cost-effective, integrative analysis of transcriptome and paired BCR or TCR repertoire in phenotypically defined B and T cell subsets. They describe in detail the experimental workflow and provide a robust bioinformatics pipeline for computing gene count matrices and reconstructing repertoire sequences from FB5P-seq data. The researchers further present two applications of FB5P-seq for the analysis of human tonsil B cell subsets and peripheral blood antigen-specific CD4 T cells. They believe that our novel integrative scRNA-seq method will be a valuable option to study rare adaptive immune cell subsets in immunology research.

Overview of the FB5P-seq experimental workflow

rna-seq

(A) Major experimental steps of the FB5P-seq workflow. (B) Schematic overview of the molecular designs and the reactions in the FB5P-seq workflow. (C) Schematic illustration of the mapping of the Read1 sequences (in red) on IGH and IGK or IGL amplified cDNA, enabling the in silico reconstruction of paired variable BCR sequences.

Attaf N, Cervera-Marzal I, Dong C, Gil L, Renand A, Spinelli L, Milpied P. (2020) FB5P-seq: FACS-Based 5-Prime End Single-Cell RNA-seq for Integrative Analysis of Transcriptome and Antigen Receptor Repertoire in B and T Cells. Front Immunol 11:216. [article]

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