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Targeted TCR Amplification from Single-Cell cDNA Libraries.

Shuqiang Li1,2, Kenneth J Livak3

  • 1Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|April 28, 2019
PubMed
Summary
This summary is machine-generated.

This study presents a sensitive protocol for amplifying T cell receptor (TCR) genes from single cells. The method uses RNase H-dependent PCR (rhPCR) for efficient TCR allele amplification and cell barcoding in one step.

Keywords:
Paired TCRαβ single-cell sequencingRNase H-dependent PCRT cell receptor repertoire

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Area of Science:

  • Immunology
  • Molecular Biology
  • Genomics

Background:

  • T cell specificity is crucial for adaptive immunity.
  • Determining T cell receptor (TCR) alleles is key to understanding T cell specificity.
  • Existing methods for TCR sequencing can be complex and time-consuming.

Purpose of the Study:

  • To develop a sensitive and efficient protocol for targeted amplification of TCR CDR3 regions from single-cell cDNA libraries.
  • To enable accurate determination of T cell specificity through TCR allele sequencing.
  • To streamline the process of TCR analysis in single cells.

Main Methods:

  • Single-cell full-length cDNA library preparation.
  • Targeted amplification of TCR CDR3 regions.
  • Utilizing RNase H-dependent PCR (rhPCR) for allele-specific amplification and cell barcoding in a single step.

Main Results:

  • A sensitive protocol for TCR CDR3 region amplification was established.
  • The rhPCR-based method efficiently amplifies TCR alleles.
  • Cell barcodes are successfully added during the single PCR amplification step.

Conclusions:

  • The described protocol offers a sensitive and streamlined approach for single-cell TCR sequencing.
  • This method facilitates the determination of T cell specificity by enabling efficient TCR allele analysis.
  • The rhPCR strategy simplifies the workflow for comprehensive T cell repertoire analysis.