RESUMO
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.
Assuntos
Linfócitos do Interstício Tumoral/metabolismo , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Separação Celular/métodos , Cromo/química , DNA Complementar/genética , Emulsões/química , Desenho de Equipamento , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Genômica/instrumentação , Genômica/métodos , Humanos , Indicadores e Reagentes , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de RNA/instrumentação , Análise de Célula Única/instrumentaçãoRESUMO
The tumor microenvironment restrains conventional T cell (Tconv) activation while facilitating the expansion of Tregs. Here we showed that Tregs' advantage in the tumor milieu relies on supplemental energetic routes involving lipid metabolism. In murine models, tumor-infiltrating Tregs displayed intracellular lipid accumulation, which was attributable to an increased rate of fatty acid (FA) synthesis. Since the relative advantage in glucose uptake may fuel FA synthesis in intratumoral Tregs, we demonstrated that both glycolytic and oxidative metabolism contribute to Tregs' expansion. We corroborated our data in human tumors showing that Tregs displayed a gene signature oriented toward glycolysis and lipid synthesis. Our data support a model in which signals from the tumor microenvironment induce a circuitry of glycolysis, FA synthesis, and oxidation that confers a preferential proliferative advantage to Tregs, whose targeting might represent a strategy for cancer treatment.