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

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 TCRß 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.

Analysis of Transcriptional Profiling of Immune Cells at the Single-Cell Level.

RNA sequencing has proven to be a key innovation for the study of biological processes by enabling scientists to measure differences in gene expression in different tissues.With recent advances in sequencing technology, researchers are able to measure gene transcription at the single-cell level, revealing previously unknown diversity and specificity of immune cells. The single-cell sequencing method now enables profiling of the T-cell receptor (TCR) genes resulting from V(D)J recombination.Here we describe how to adapt single-cell RNA sequencing data generated using the 10× genomics 5'V(D)J immune cell profiling workflow for integration into the R analysis pipeline.We will start with the data matrix files generated from the 10× genomics Cell Ranger alignment software and detail how to format this data as input for the R analysis package called Seurat such that data from both the overall cell transcript abundance and the targeted V(D)J transcript abundance data can be visualized on the same plots.