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 >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.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
McDaniel JR,DeKosky BJ,Tanno H,Ellington AD,Georgiou Gdoi
10.1038/nprot.2016.024subject
Has Abstractpub_date
2016-03-01 00:00:00pages
429-42issue
3eissn
1754-2189issn
1750-2799pii
nprot.2016.024journal_volume
11pub_type
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