Benchmarking Single-Cell mRNA–Sequencing Technologies Uncovers Differences in Sensitivity and Reproducibility in Cell Types With Low RNA Content

Abstract

Single-cell RNA sequencing (scRNA-seq) has the ability to classify each cell and determine the transcriptomic profile of specific cell types and cells of a given disease state; however, sensitivity of the gene count for each cell can be a critical component to the success of a single-cell study. The recently introduced SMART-Seq Single Cell PLUS Kit (SSsc PLUS) claims to provide higher sensitivity and reproducibility versus popular methods for the sequencing analysis of single cells. Here, the cDNAgeneration component of the kit, SMART-Seq Single Cell Kit (SSsc), was compared with the popular homebrew protocol, Smart-seq2, and its update, Smart-seq3. The SMART-Seq Library Prep Kit from SSsc PLUS was benchmarked against a commonly used scRNA-seq library preparation method, Illumina Nextera XT. Finally, the SSsc chemistry was tested in both full and fractional volumes on 2 popular liquid-handler devices to investigate whether the high sensitivity was maintained in miniaturization.We demonstrate that SSsc PLUS outperforms these other full-length methods in convenience, sensitivity, gene identification, and reproducibility while also offering full compatibility with automation platforms.