Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy.
Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
Methods Mol Biol. 2021;2285:277-296. doi: 10.1007/978-1-0716-1311-5_22.
During the last decade, the rapid progress in the development of next-generation sequencing (NGS) technologies has provided relevant insights into complex biological systems, ranging from cancer genomics to microbiology. Among NGS technologies, single-cell RNA sequencing is currently used to decipher the complex heterogeneity of several biological samples, including T cells. Even if this technique requires specialized equipment and expertise, nowadays it is broadly applied in research. In this chapter, we will provide an optimized protocol for the isolation of T cells and the preparation of RNA sequencing libraries by using droplet digital technology (ddSEQ, Bio-Rad Laboratories). We will also illustrate a guide to the main steps of data processing and options for data interpretation. This protocol will support users in building a single-cell experimental framework, from sample preparation to data interpretation.
在过去的十年中，下一代测序（NGS）技术的快速发展为复杂的生物系统提供了相关的见解，范围从癌症基因组学到微生物学。在 NGS 技术中，单细胞 RNA 测序目前用于破译包括 T 细胞在内的几种生物样本的复杂异质性。即使这项技术需要专门的设备和专业知识，如今它也被广泛应用于研究中。在本章中，我们将提供一种优化的方案，用于使用液滴数字技术（ddSEQ，Bio-Rad Laboratories）分离 T 细胞和制备 RNA 测序文库。我们还将说明主要数据处理步骤和数据解释选项的指南。本方案将支持用户从样品制备到数据解释构建单细胞实验框架。
