RNA-sequencing (RNA-seq) is a powerful technique to investigate and quantify entire transcriptomes. Recent advances in the field have made it possible to explore the transcriptomes of single cells. However, most widely used RNA-seq protocols fail to provide crucial information regarding transcription start sites. Here we present a protocol, Tn5Prime, that takes advantage of the Tn5 transposase-based Smart-seq2 protocol to create RNA-seq libraries that capture the 5′ end of transcripts. The Tn5Prime method dramatically streamlines the 5′ capture process and is both cost effective and reliable. By applying Tn5Prime to bulk RNA and single cell samples, we were able to define transcription start sites as well as quantify transcriptomes at high accuracy and reproducibility. Additionally, similar to 3′ end-based high-throughput methods like Drop-seq and 10× Genomics Chromium, the 5′ capture Tn5Prime method allows the introduction of cellular identifiers during reverse transcription, simplifying the analysis of large numbers of single cells. In contrast to 3′ end-based methods, Tn5Prime also enables the assembly of the variable 5′ ends of the antibody sequences present in single B-cell data. Therefore, Tn5Prime presents a robust tool for both basic and applied research into the adaptive immune system and beyond.

中文翻译：

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Tn5Prime，一种基于 Tn5 的单细胞 RNA-seq 5' 捕获方法

RNA 测序 (RNA-seq) 是一种研究和量化整个转录组的强大技术。该领域的最新进展使得探索单细胞的转录组成为可能。然而，最广泛使用的 RNA-seq 协议无法提供有关转录起始位点的关键信息。在这里，我们提出了一个协议 Tn5Prime，它利用基于 Tn5 转座酶的 Smart-seq2 协议来创建捕获转录本 5' 端的 RNA-seq 文库。Tn5Prime 方法极大地简化了 5' 捕获过程，并且既经济又可靠。通过将 Tn5Prime 应用于大量 RNA 和单细胞样本，我们能够以高精度和可重复性定义转录起始位点以及量化转录组。此外，类似于基于 3' 端的高通量方法，如 Drop-seq 和 10× Genomics Chromium，5' 捕获 Tn5Prime 方法允许在逆转录过程中引入细胞标识符，简化大量单细胞的分析。与基于 3' 端的方法相比，Tn5Prime 还能够组装单个 B 细胞数据中存在的抗体序列的可变 5' 端。因此，Tn5Prime 为适应性免疫系统及其他领域的基础研究和应用研究提供了强大的工具。Tn5Prime 还能够组装单个 B 细胞数据中存在的抗体序列的可变 5' 端。因此，Tn5Prime 为适应性免疫系统及其他领域的基础研究和应用研究提供了强大的工具。Tn5Prime 还能够组装单个 B 细胞数据中存在的抗体序列的可变 5' 端。因此，Tn5Prime 为适应性免疫系统及其他领域的基础研究和应用研究提供了强大的工具。