Enriching and Characterizing T-Cell Repertoires from 3' Barcoded Single-Cell Whole Transcriptome Amplification Products
| Autor: | Tasneem Jivanjee, Samira Ibrahim, Sarah K. Nyquist, G. James Gatter, Joshua D. Bromley, Swati Jaiswal, Bonnie Berger, Samuel M. Behar, J. Christopher Love, Alex K. Shalek |
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| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: | |
| Zdroj: | Methods Mol Biol Methods in Molecular Biology ISBN: 9781071627112 |
| Popis: | Antigen-specific T cells play an essential role in immunoregulation and 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, requiring 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, it requires specialized reagents which cannot be applied to samples previously processed using 3' cell-barcoding methods. Here, we outline a method for sequencing TCR$\alpha$ and TCR$\beta$ 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 results 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. This method can be adapted to enrich other transcripts of interest from 3' and 5' barcoded WTA libraries. Comment: 57 pages excluding supplementary figures, 7 figures, 8 tables, 5 supplementary figures |
| Databáze: | OpenAIRE |
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