FB5P-seq: FACS-based 5-prime end single-cell RNAseq for integrative analysis of transcriptome and antigen receptor repertoire in B and T cells

Single-cell RNA sequencing (scRNAseq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on B and T cells of the adaptive immune system, scRNAseq 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. Here we introduce FB5Pseq, a FACS-based 5’-end scRNAseq method for cost-effective integrative analysis of transcriptome and paired BCR or TCR repertoire in phenotypically defined B and T cell subsets. We describe in details the experimental workflow and provide a robust bioinformatics pipeline for computing gene count matrices and reconstructing repertoire sequences from FB5Pseq data. We further present two applications of FB5Pseq for the analysis of human tonsil B cell subsets and peripheral blood antigen-specific CD4 T cells. We believe our novel integrative scRNAseq method will be a valuable option to study rare adaptive immune cell subsets in immunology research. This dataset contains data from B cell subsets sorted from two non-malignant human tonsil samples (Tonsil 1 and Tonsil 2), and C.alb-specific T cells sorted from human peripheral blood after in vitro peptide restimulation. Single-cells were FACS-sorted into several 96-well plates (Tonsil 1: 5 plates; Tonsil 2: 6 plates; T cells: 1 plate) and analyzed by 5'-end single-cell RNAseq following the FB5P-seq protocol. The three samples (Tonsil 1, Tonsil 2, T cells) were processed and sequenced in three separate experiments.