| Abstract [eng] |
Single-cell IgG Gene Amplification Using Droplet-based Microfluidics Characterization of essential players in the immune system – antibodies (immunoglobulins) – is of key importance in the understanding of adaptive immune responses in healthy and diseased patients. However, sequencing analysis of both antibody chains at the single-cell level for typical samples containing more than ten thousands lymphocytes is problematic, because antibodies are comprised of two polypeptide chains that are encoded by separate mRNAs. To overcome those difficulties, here we present a technology that allows fast, reliable and low-cost determination of a paired antibody repertoire from millions of B cells. Droplet-based microfluidic is used to encapsulate single cells in emulsions containing hydrogel beads with poly-T primers and lysis-reverse transcription reagents for mRNA capture and cDNA synthesis. After reverse transcription cDNA molecules are covalently linked via poly-T primers with hydrogel beads that are re-encapsulated to perform overlap-extension PCR. VH:VL pairs obtained during overlap extension PCR are pooled together and analyzed using next generation sequencing techniques. |
