Journal
NATURE PROTOCOLS
Volume 11, Issue 3, Pages 429-442Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.024
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Funding
- Defense Threat Reduction Agency [HDTRA1-12-C-0105]
- National Institutes of Health (NIH) [R01AI096228]
- Hertz Foundation
- University of Texas Donald D. Harrington Foundation
- National Science Foundation
- Japan Society for the Promotion of Science (JSPS)
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High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCR beta/delta+TCR alpha/gamma) at the single-cell level for typical samples containing > 10(4) lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (> 97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample.
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