Chief Scientific Officer Cellecta, California, United States
Disclosure(s):
Alex Chenchik, PhD: No relevant disclosure to display
Introduction/Rationale: 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.
