Chromium 10x Single-Cell 3′ mRNA Sequencing of Tumor-Infiltrating Lymphocytes
Chromium 10x Single-Cell 3′ mRNA Sequencing of Tumor-Infiltrating Lymphocytes
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DOI：
10.1007/978-1-4939-9240-9_7
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发表时间：
2019-01-01
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影响因子：
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通讯作者：
Pagani, Massimiliano
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文献类型：
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作者：
De Simone, Marco;Rossetti, Grazisa;Pagani, Massimiliano
铬10x 3‘V2法是一种3’端计数的单细胞mRNA测序方法，允许并行处理和测序来自数千个细胞的RNA。Chromium10x by 10x基因组学是一种基于乳液的设备，它能够将单细胞与一组独特的条形码引物和逆转录试剂分隔成纳米级的液滴，这些液滴用作反应室，从单细胞中生成条形码的全长DNA。RT反应后，将单链条码cDNA汇集在一起并进行处理，以产生与标准Illumina平台兼容的测序文库。在这里，我们详细地展示了应用于肿瘤浸润性T淋巴细胞(TIL)分析的主要步骤。主要步骤是细胞制备、基因合成、文库构建和测序。本协议专门针对可从10x基因组学网站(CG00052_SingleCell3_ReagentKitv2UserGuide_RevD)下载的https://www.10xgenomics.com)，并且不替代它。请始终参考本指南，注意更新和修订。
Chromium 10x 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. Chromium10x by 10x 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 10x Genomics website (https://www.10xgenomics.com) and does not substitute it. Always refer to this guide, paying attention to updates and revisions.