Publication Date:
2013-02-08
Description:
Nature Biotechnology 31, 166 (2013). doi:10.1038/nbt.2492 Authors: Brandon J DeKosky, Gregory C Ippolito, Ryan P Deschner, Jason J Lavinder, Yariv Wine, Brandon M Rawlings, Navin Varadarajan, Claudia Giesecke, Thomas Dörner, Sarah F Andrews, Patrick C Wilson, Scott P Hunicke-Smith, C Grant Willson, Andrew D Ellington & George Georgiou 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 (VH and VL) 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 × 104 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 VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL 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.
Print ISSN:
1087-0156
Electronic ISSN:
1546-1696
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
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