NET-Seq
TCR Chain Pairing
This method identifies TCR-alpha and -beta chain pairing in single cells using cell-based emulsion technology for isolation, followed by NGS. TCR chain pairing resolves one of the biggest challenges of identifying coexpressed gene pairs—random, nonspecific overlap extension of nonfused molecules—by introducing a unique PCR suppression technique during post–emulsion amplification reactions. First, single T cells are isolated into oil emulsion droplets containing RT primers for alpha and beta chain mRNA strands. The resultant cDNA is amplified, and the RT primer extensions overlapped to connect TCR-alpha and TCR-beta strands, which are now called fused molecules. The cDNA products are extracted from the emulsion and introduced to blocking primers. These primers anneal to the 3' end of nonfused cDNA strands, preventing them from being amplified; the authors name this technique “PCR suppression.” Finally, the fused molecules are PCR-amplified and sequenced.
Similar methods: TCR-LA-MC PCR
Pros:
- Identifies TCR chain pairing using NGS
- PCR suppression of nonfused molecules significantly reduces random, nonspecific overlap extension during post–emulsion amplification reactions
- Simple and straightforward protocol
Cons:
- Heat-shock during cell lysis may reduce enzymatic activity during RT1
- Amplification product is not suitable for cloning2
- TCR Chain Pairing: Turchaninova M. A., Britanova O. V., Bolotin D. A., et al. Pairing of T-cell receptor chains via emulsion PCR. Eur J Immunol. 2013;43:2507-2515
- 1. Friedensohn S., Khan T. A. and Reddy S. T. Advanced Methodologies in High-Throughput Sequencing of Immune Repertoires. Trends in Biotechnology. 2017;35:203-214
- 2. Sprouse M. L., Blahnik G., Lee T., et al. Rapid identification and expression of human TCRs in retrogenic mice. J Immunol Methods. 2016;