Abstract LB-078: Improved single-cell mRNA sequencing for transcriptome and paired-chain TCR analysis of primary human CD3+ T cells
Authors: Egidio Camila , Gonzales Michael , Brockman Joel , Chen Shuwen , Durruthy-Durruthy Robert , Rogers Clare , Ray Manisha 
Published: July 2017

The low RNA content of single human T cells has limited the success of single-cell transcriptome profiling, an approach that can generate data to reconstruct paired α,β T cell receptor (TCR) chains¹. Single-cell TCR sequencing is a powerful molecular tool to analyze tumor-infiltrating lymphocytes and production of cellular therapeutics. We compared the sensitivity and overall performance of the new Clontech® SMART-Seq® v4 Kit to the previously validated SMART-Seq v1 Kit on single primary human CD3+ cells captured and processed on the C1™ system (Fluidigm®). Sequenced libraries were analyzed using the TraCeR bioinformatics pipeline¹ to identify the TCR α and β chains expressed in each T cell. Cryopreserved CD3+ T cells were prepared as a single-cell suspension for capture on C1 using SMART-Seq v1 and v4, according to manufacturer instructions (AllCells®, Fluidigm, Clontech). Single-cell libraries were sequenced at 1.9 million reads per single cell. After data processing, the aligned reads were analyzed using the TraCeR bioinformatics pipeline. Transcriptome-related metrics and paired-chain TCR information were compared to evaluate chemistry performance (Table 1). Results are comparable to single-cell data published by Clontech for cultured Jurkat cells², which do not pose as high a technical challenge to molecular profiling as the primary T cells used in this study. The v4 chemistry produced a higher number of single cells with paired α,β chains detected. These results show that combining the C1 system with SMART-Seq v4 chemistry for processing primary single human T cells provides a powerful tool to investigate and enumerate T cell clones and their phenotypes and functions. 1. Stubbington, M.J.T. et al. Nature Methods, 2016. 2. Bostick, M. et al. Poster, NIH Single Cell Analysis Investigators Meeting, 2016.

Table 1.Metrics evaluated in the comparison of SMART-Seq v1 and v4 chemistry performance.MetricSMART-Seq v1 (98 cells)SMART-Seq v4 (122 cells)cDNA yield per single cell (ng)3.4 (±1.4)4.9 (±2.0)Reads aligned to the genome from total (%)82 (±3.2)77 (±6.4)Reads with unique alignment to the genome from total aligned (%)97 (±0.04)97 (±0.05)Reads aligned to RefSeq (%)38 (±0.7)56 (±0.06)Reads aligned to rRNA (%)0.61 (±0.03)1.31 (±0.05)Bases aligned to coding sequences (%)22 (±0.5)40 (±0.5)Bases aligned to intronic sequences (%)40 (±1.0)18 (±0.5)Genes detected per T cell in average (above 1 TPM)2,618 (±431)1,866 (±386)Number of cells for which paired α,β T cell receptor (TCR) chains were identified25 (25%)67 (55%)

Citation Format: Camila Egidio, Michael Gonzales, Joel Brockman, Shuwen Chen, Robert Durruthy-Durruthy, Clare Rogers, Manisha Ray. Improved single-cell mRNA sequencing for transcriptome and paired-chain TCR analysis of primary human CD3+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-078. doi:10.1158/1538-7445.AM2017-LB-078