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.

Noudjoud Attaf,Amédée Renand,Laurine Gil,Chuang Dong,Iñaki Cervera-Marzal,Pierre Milpied,Lionel Spinelli

doi:10.3389/fimmu.2020.00216

Abstract

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. Here we 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. We 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. We further present two applications of FB5P-seq for the analysis of human tonsil B cell subsets and peripheral blood antigen-specific CD4 T cells. We believe that our novel integrative scRNA-seq method will be a valuable option to study rare adaptive immune cell subsets in immunology research.

Highlights

Technologies to reliably amplify and sequence the mRNA content of single cells have progressed dramatically
Single cells are collected in lysis buffer containing External RNA Controls Consortium (ERCC) spikein mRNA (0.025 pg per well), and the sorted plates are immediately frozen on dry ice and stored at −80◦C until further processing
The amount of ERCC spike-in mRNA added to each well was optimized to yield around 5% of sequencing reads covering ERCC genes when studying lymphocytes which generally contain little mRNA. mRNA reverse transcription, cDNA 5′-end barcoding, and PCR amplification are performed with a template switching (TS) approach

Summary

Introduction

Technologies to reliably amplify and sequence the mRNA content of single cells have progressed dramatically. By quantitatively measuring the expression levels of thousands of genes per cell, single-cell RNA sequencing (scRNA-seq) enables an unbiased classification of cell types and fine characterization of functional cell states [1, 2]. All scRNA-seq protocols are based on four common steps: (i) single cell isolation, (ii) reverse transcription (RT) of mRNA, (iii) amplification of cDNA, and (iv) preparation of next-generation sequencing libraries. FACS has the advantage of allowing the user to record the precise cell surface phenotype of each sorted cell (index sorting) and link it to its deeply sequenced transcriptome (>2,000 genes/cell), but with a limited throughput of a few hundred cells per sample [8, 9]

Methods

Results

Conclusion