Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes

JR McDaniel, BJ DeKosky, H Tanno, AD Ellington… - Nature protocols, 2016 - nature.com
Nature protocols, 2016nature.com
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 TCRβ/δ+ TCRα/γ) at the single-cell level for typical samples containing> 104
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 …
Abstract
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 TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >104 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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