Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy-and light-chain variable regions (V H and V L) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10 4 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V H:V L linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V H:V L pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG + B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology
- Molecular Medicine
- Biomedical Engineering