Abstract
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.