De Simone Marco, Rossetti Grazisa, Pagani Massimiliano
Istituto Nazionale Genetica Molecolare INGM 'Romeo ed Enrica Invernizzi', Milan, Italy.
Department of Medical Biotechnology and Translational Medicine, Università Degli Studi di Milano, Milan, Italy.
Methods Mol Biol. 2019;1979:87-110. doi: 10.1007/978-1-4939-9240-9_7.
Chromium 10× 3' V2 protocol is a 3' end counting single-cell mRNA sequencing protocol that allows to process and sequence RNA from thousands of cells in parallel. Chromium10× by 10× Genomics is an emulsion-based device that enables to compartmentalize single cells along with sets of uniquely barcoded primers and reverse transcription reagents into nanoscale droplets that are used as reaction chambers to generate barcoded full-length cDNA from single cells. After RT reaction single-stranded barcoded cDNAs are pooled together and processed to generate sequencing libraries compatible with the standard Illumina platforms. Here we show in detail the main steps of the protocol applied to the analysis of tumor-infiltrating T lymphocytes (TILs). The main steps are cell preparation, cDNA synthesis, library construction, and sequencing.This protocol refers specifically to the CG00052_SingleCell3_ReagentKitv2UserGuide_RevD downloadable from 10× Genomics website ( https://www.10xgenomics.com ) and does not substitute it. Always refer to this guide, paying attention to updates and revisions.
铬10×3'V2协议是一种3'端计数单细胞mRNA测序协议，可并行处理和测序来自数千个细胞的RNA。10×基因组学公司的铬10×是一种基于乳液的设备，能够将单细胞与一组独特的条形码引物和逆转录试剂一起分隔到纳米级液滴中，这些液滴用作反应室，从单细胞生成条形码全长cDNA。RT反应后，单链条形码cDNA被汇集在一起并进行处理，以生成与标准Illumina平台兼容的测序文库。在这里，我们详细展示了应用于肿瘤浸润性T淋巴细胞（TILs）分析的该协议的主要步骤。主要步骤包括细胞制备、cDNA合成、文库构建和测序。本协议具体参考了可从10×基因组学网站（https://www.10xgenomics.com）下载的CG00052_SingleCell3_ReagentKitv2UserGuide_RevD，并不替代该指南。请始终参考本指南，并留意更新和修订内容。
