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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Acknowledgements
We thank A. Kennedy for his assistance in making the borosilicate glass nozzles. Funding for this work was provided by Defense Threat Reduction Agency HDTRA1-12-C-0105 (G.G.), National Institutes of Health (NIH) Grant R01AI096228 (G.G.), and fellowships to B.J.D. from the Hertz Foundation, the University of Texas Donald D. Harrington Foundation and the National Science Foundation. H.T. was supported by Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research Abroad.
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J.R.M., B.J.D. and G.G. wrote the manuscript. J.R.M., B.J.D., H.T., A.D.E and G.G. designed the experiments. J.R.M., B.J.D. and H.T. performed the experiments and conducted bioinformatic analyses. All authors edited and approved the manuscript.
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G.G., B.J.D. and A.D.E. declare competing financial interests in the form of a patent filed by the University of Texas at Austin.
Integrated supplementary information
Supplementary Figure 1 Alignment of custom-designed glass nozzle.
A) PBS/PBS aqueous phases in oil; nozzle aperture is properly aligned. B) PBS/Lysis Buffer aqueous phases in oil; nozzle aperture is properly aligned. The presence of surfactants greatly reduces the width of the aqueous stream. C) PBS/Lysis Buffer aqueous phases in oil; aperture is off-center. D) PBS/Lysis Buffer aqueous phases in oil; aperture is so far off-center that the aqueous phase is coming into contact with the curvature of the glass nozzle. In all pictures, the stream is flowing right to left.
Supplementary Figure 2 Emulsions collected from flow-focusing device before and after centrifugation.
The top layer of mineral oil containing microfines (small emulsions) should be removed and discarded. The bottom layer of larger emulsions contains cells and beads, which should be retained. The color will change depending on the concentration of the poly(dT) beads.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1 and 2, Supplementary Table 1 (PDF 476 kb)
Aqueous/oil interface for misaligned glass nozzle.
If the concentric metal tubes are not centered on the aperture of the nozzle, the aqueous stream will flicker or appear asymmetric. If the needle-to-aperture distance is too great (>2-3 mm), the aqueous stream will flicker and may form a second node. (MOV 2845 kb)
Aqueous/oil interface during the transition from the PBS/PBS aqueous phase to the PBS/Lysis Buffer aqueous phase.
The introduction of surfactant into the aqueous phase greatly reduces the diameter of the stream. The transition is also typically accompanied by air bubbles that are introduced while switching syringes. Sample collection should begin once the bubbles cease and the stream becomes stable. (MOV 1053 kb)
Supplementary Methods
Bioinformatics script written in Python to parse and pair the VH and VL sequences following IMGT gene annotation. (TXT 17 kb)
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McDaniel, J., DeKosky, B., Tanno, H. et al. Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes. Nat Protoc 11, 429–442 (2016). https://doi.org/10.1038/nprot.2016.024
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DOI: https://doi.org/10.1038/nprot.2016.024
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