Paper

Enriching and Characterizing T-Cell Repertoires from 3' Barcoded Single-Cell Whole Transcriptome Amplification Products

Nyquist, Sarah K, Ibrahim, Samira, Jivanjee, Tasneem, Shalek, Alex K, Behar, Samuel M, Bromley, Joshua D, Jaiswal, Swati, Love, J Christopher, Berger, Bonnie, Gatter, G James

2022

Overview

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.

• Publisher: Cornell University Library, arXiv.org
• Subject: Amplification / Antigens / Enrichment / Gene sequencing / Heterogeneity / Immunology / Libraries / Lymphocytes / Reagents / T cell receptors