Abstract
Antigen-specific T cells play an essential role in immunoregulation and many diseases such as cancer. Characterizing the T cell receptor (TCR) sequences that encode T cell specificity is critical for elucidating the antigenic determinants of immunological diseases and designing therapeutic remedies. However, methods of obtaining single-cell TCR sequencing data are labor and cost intensive, typically requiring both cell sorting and full-length single-cell RNA-sequencing (scRNA-seq). New high-throughput 3′ cell-barcoding scRNA-seq methods can simplify and scale this process; however, they do not routinely capture TCR sequences during library preparation and sequencing. While 5′ cell-barcoding scRNA-seq methods can be used to examine TCR repertoire at single-cell resolution, doing so requires specialized reagents which cannot be applied to samples previously processed using 3′ cell-barcoding methods.
Here, we outline a method for sequencing TCRα and TCRβ transcripts from samples already processed using 3′ cell-barcoding scRNA-seq platforms, ensuring TCR recovery at a single-cell resolution. In short, a fraction of the 3′ barcoded whole transcriptome amplification (WTA) product typically used to generate a massively parallel 3′ scRNA-seq library is enriched for TCR transcripts using biotinylated probes and further amplified using the same universal primer sequence from WTA. Primer extension using TCR V-region primers and targeted PCR amplification using a second universal primer result in a 3′ barcoded single-cell CDR3-enriched library that can be sequenced with custom sequencing primers. Coupled with 3′ scRNA-seq of the same WTA, this method enables simultaneous analysis of single-cell transcriptomes and TCR sequences which can help interpret inherent heterogeneity among antigen-specific T cells and salient disease biology. The method presented here can also be adapted readily to enrich and sequence other transcripts of interest from both 3′ and 5′ barcoded scRNA-seq WTA libraries.
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1 Electronic Supplementary Material
Supplementary Table 1
Example output of TCRGO pipeline (TSV 51 kb)
Supplementary Table 2
Biotinylated oligonucleotide probes for TCR enrichment for human (Homo sapiens), mouse (Mus musculus), and crab-eating macaque (Macaca fascicularis, abbreviated MacFas), respectively (CSV 1 kb)
Supplementary Table 3
UPS2_N50× primers with Illumina barcodes (CSV 5 kb)
Supplementary Table 4
Optimized custom TCR sequencing primers for human, mouse, and crab-eating macaque. Human/macaque primers are universal for human and macaque species (CSV 574 bytes)
Supplementary Table 5
(a–c) Species-specific variable region primers: (a) human, (b) mouse, and (c) crab-eating macaque (MacFas) (CSV 6 kb)
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Jivanjee, T. et al. (2022). Enriching and Characterizing T Cell Repertoires from 3′ Barcoded Single-Cell Whole Transcriptome Amplification Products. In: Huang, H., Davis, M.M. (eds) T-Cell Repertoire Characterization. Methods in Molecular Biology, vol 2574. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2712-9_7
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DOI: https://doi.org/10.1007/978-1-0716-2712-9_7
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