Streamlined and cost-effective 96-well plate NGS assay for single-cell immune receptor chain pairing and T/B cell immunophenotyping 2260349

Abstract

Abstract Introduction To facilitate the discovery of epitopes or immune therapy targets for researchers without ready access to expensive instrumentation or reagents, we present a cost-effective, simplified workflow for single-cell immune receptor (TCR/BCR) profiling. Methods We use a standard 96-well plate; T- or B-cells are flow-sorted into wells, primers for immune receptor and key marker genes are added, followed by multiplex RT-PCR and next-generation sequencing (NGS). Novel validator barcodes (VBCs) ensure robust clonotype quantification with minimal cross-well contamination. There are two workflow options: one can either 1) flow-sort 96 individual cells onto a plate, or 2) serially dilute ∼10,000 cells (∼100 cells/well in a 96-well plate), which allows for thorough characterization of each receptor in larger cell populations. Results Data analysis with MiXCR software yields full-length receptor chain-pair sequences, clonotype abundance, and gene expression profiles. Single-cell results show 86% of the sorted cells had an associated clonotype, 65% had a TCR-αβ chain pair. Conclusion This sensitive assay is ideal for working with rare or enriched cell populations of a few thousand cells. The single-day workflow, which includes pooling of a 96-well plate into one reaction and profiling multiple plates in one batch, drops assay costs to ∼10 cents/cell. The approach will facilitate applications such as engineering T-cell-based therapies, antibody synthesis, and epitope discovery for cancer or autoimmune diseases. Funding Source N/A Topic Categories Technological Innovations in Immunology (TECH)