TRACE-seq: “TRanslatomic” Analysis Captured in Extracellular vesicles using sequencing. TRACE-seq: “TRanslatomic” Analysis Captured in Extracellular vesicles using sequencing

Purpose: We develop a new methodology to monitor live cells in vitro as well as in vivo. Method: Each population of HEK 293T cells was transfected with the TRACE-seq construct (GFP-C-YTHDF1/GBP1-CD9) and a negative control construct (GFP-C-YTHDF1/GBP1-CD9) which contains no mRNA catcher. The analysis was made in duplicate with microvesicles (MVs) mRNA content isolated from cell culture media and their corresponding cell lysate. Samples were generated with the SMART-seq2 protocol and the Nextera kit (Illumina). Data were collected using 50 × 8 × 50 reads on a HiSeq. Reads were aligned to hg19 using STAR and counting reads associated genes were detected with the FeatureCounts module. Also, a QC qPCR was made on these samples and the cDNA library quality was analyzed by a High sensitivity DNA chip assay. Results: From the 3604 genes selected for this analysis, RNA-seq data showed a non negligeable increase of the correslation between MVs mRNA content and cell lysate from the HEK293T transfected with the TRACE construct. Conclusion: Our methodology bring a representative part of the transcriptome from the cell into MVs which is a usefull method for in vivo analysis. Moreover, our study represents the first detailed analysis of a new undestructive, fully compatible for in vivo, monitoring process of the cells transcriptome. Overall design: HEK 293T cells transfected with two construct: TRACE one (GFP-C-YTHDF1/GBP1-CD9) and the negatif control construct (GFP/GBP1-CD9). Analyses made on microvesicles from the cells media and the own cells lysate of each samples.

研究目的：本研究旨在开发一种可同时用于体外（in vitro）与活体内（in vivo）活细胞监测的新型方法学。
实验方法：将每批HEK 293T细胞（HEK 293T cells）分别转染TRACE-seq构建体（TRACE-seq construct，GFP-C-YTHDF1/GBP1-CD9）以及不含mRNA捕获元件（mRNA catcher）的阴性对照构建体（GFP-C-YTHDF1/GBP1-CD9）。本实验对从细胞培养基中分离得到的微囊泡（microvesicles, MVs）mRNA组分及其对应细胞裂解液开展了重复实验分析。测序文库采用SMART-seq2建库流程（SMART-seq2 protocol）与Illumina公司的Nextera建库试剂盒（Nextera kit）制备。测序数据在HiSeq测序平台（HiSeq）上以50×8×50的读长参数进行采集。使用STAR比对工具（STAR）将测序读段比对至hg19参考基因组（hg19），随后通过FeatureCounts模块（FeatureCounts module）完成计数关联基因的检测。此外，对上述样本开展了质控定量PCR（quantitative PCR, qPCR）实验，并采用高灵敏度DNA芯片分析法对cDNA文库（cDNA library）的质量进行了评估。
实验结果：在本次分析选取的3604个基因中，RNA测序数据显示，转染TRACE-seq构建体的HEK 293T细胞，其微囊泡mRNA组分与对应细胞裂解液的转录组相关性呈现显著提升。
研究结论：本研究开发的方法学可将细胞转录组的代表性组分载入微囊泡中，为活体内分析提供了一种有效的研究手段。此外，本研究首次针对一种全新的非破坏性、完全适配活体内环境的细胞转录组监测流程开展了详细分析。
实验整体设计：将HEK 293T细胞分为两组，分别转染TRACE-seq构建体（GFP-C-YTHDF1/GBP1-CD9）与阴性对照构建体（GFP/GBP1-CD9）；后续分析分别针对各组样本的细胞培养基来源微囊泡及其对应细胞裂解液展开。