GSE162425
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.