title: Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes.
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.
authors: Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
authors: Jonathan R
authors: http://orcid.org/0000-0001-8575-3535
authors: JR
authors: McDaniel
authors: Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.
authors: Brandon J
authors: BJ
authors: DeKosky
authors: Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
authors: Hidetaka
authors: H
authors: Tanno
authors: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.|Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, USA.
authors: Andrew D
authors: AD
authors: Ellington
authors: Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.|Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.
authors: George
authors: G
authors: Georgiou
mesh_terms: D004867:Equipment Design; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D056656:Lab-On-A-Chip Devices / Q000191:economics; D008214:Lymphocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*
chemical_list: D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell