RNA-seq identifies a distinct response in macrophages stimulated with early secreted antigenic target 6-KDa from Mycobacterium tuberculosis. RNA-seq identifies a distinct response in macrophages stimulated with early secreted antigenic target 6-KDa from Mycobacterium tuberculosis

Purpose: The goals of this study are to obtain the NGS-derived transcriptome profiling (RNA-seq) for THP-1 macrophages response to early secreted antigenic target 6-KDa (ESAT6) from Mycobacterium tuberculosis Methods: mRNA profiles of THP-1 macrophages treated with ESAT6 were generated by deep sequencing, using Illumina Hiseq3000. The sequence reads that passed quality filters were first mapped to the latest UCSC transcript set using Bowtie2 (version 2.1.0). Then the gene expression level was estimated using RSEM (RNA-Seq by Expectation Maximization, v1.2.15), and normalized with TMM (trimmed mean of M-values) to identify differentially expressed genes (DEGs) using the edgeR package edgeR. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 23 million sequence reads per sample to the human genome (GRCh38/hg38) and identified 25,343 transcripts. Conclusions: Our study represents the first detailed analysis of transcriptomes for macrophages response to ESAT6, generated by RNA-seq technology. Overall design: NGS-derived transcriptome profiling (RNA-seq) for THP-1 macrophages response to early secreted antigenic target 6-KDa (ESAT6) from Mycobacterium tuberculosis.

研究目的：本研究旨在获取结核分枝杆菌（Mycobacterium tuberculosis）来源的早期分泌抗原靶标6kDa（early secreted antigenic target 6-KDa, ESAT6）刺激THP-1巨噬细胞后的下一代测序（Next Generation Sequencing, NGS）转录组谱（RNA-seq）数据。
研究方法：采用Illumina Hiseq3000平台开展深度测序，获取ESAT6处理的THP-1巨噬细胞的mRNA表达谱。首先将通过质量过滤的序列读段比对至最新版UCSC转录本集，比对工具为Bowtie2（版本2.1.0）；随后使用RSEM（RNA-seq期望最大化算法，v1.2.15）估算基因表达水平，通过TMM（截尾均值归一化法，trimmed mean of M-values）完成归一化，并借助edgeR软件包（edgeR）筛选差异表达基因（differentially expressed genes, DEGs）。最后采用SYBR Green荧光染料法进行实时定量PCR（qRT-PCR）验证。
研究结果：通过优化的数据分析流程，本研究将每个样本约2300万条序列读段比对至人类基因组（GRCh38/hg38），共鉴定得到25343个转录本。
研究结论：本研究为首例通过RNA-seq技术完成的巨噬细胞对ESAT6应答的转录组详细分析。
整体实验设计：针对结核分枝杆菌来源的早期分泌抗原靶标6kDa（ESAT6）刺激THP-1巨噬细胞的NGS转录组谱（RNA-seq）分析。