Single-cell sequencing of full-length transcripts and T-cell receptors with automated high-throughput Smart-seq3 [HT SS3]

We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow via robotic implementation and established best practices to consistently achieve high cell capture efficiency and data quality. In comparison with the 10X platform, HT Smart-seq3 analysis of primary CD4+ T-cells demonstrated superior sensitivity in gene detection and similar capability to capture major cellular heterogeneity upon sufficient scaling up. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified more productive pairs of alpha and beta chains without additional primer design, enabling more comprehensive profiling of TCRs. Collectively, HT Smart-seq3 provides a cost-effective and scalable method for characterization of single-cell transcriptomes and immune repertoires. PPBMCs were isolated from two healthy donors and subsequently treated with either DMSO (vehicle) or PMA/Ionomycin. After a 3-hour incubation, FACS-sorted CD4+ T-cells were collected for single-cell library preparation using the HT Smart-seq3 protocol.