High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S³ (“Second-Strand Synthesis”), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S³ increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S³ to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

高通量单细胞 RNA 测序 (scRNA-seq) 方法通过增加可同时分析的细胞数量来表征复杂的生物样品。然而，与低通量策略相比，这些方法在每个细胞中恢复的信息较少。为了准确报告细胞关键表型特征的表达，需要高保真度和高通量的 scRNA-seq 平台。为了满足这一需求，我们创建了 Seq-Well S ³ （“第二链合成”），这是一种大规模并行 scRNA-seq 方案，它使用随机引发的第二链合成来恢复已成功逆转录的互补 DNA (cDNA) 分子但由于模板切换效率低下，没有附加后续全转录组扩增所需的第二个寡核苷酸句柄。与之前的迭代相比，Seq-Well S ³的转录捕获和基因检测效率分别提高了 10 倍和 5 倍。我们使用 Seq-Well S ³绘制了五种人类炎症性皮肤病的转录图谱，从而为进一步研究人类皮肤炎症提供了资源。