Transcriptome analysis upon C6orf203 silencing

In our studies we were searching for the new factors engaged in mitochondrial nucleic acids metabolism under stress conditions in humans. Quantitative proteomic approach revealed C6orf203 protein as a potential new factor engaged in response to perturbed mitochondrial gene expression. We showed that C6orf203 is a mitochondrial RNA binding protein which is able to rescue diminished mitochondrial transcription in stress conditions. Overall design: The dataset corresponds to RNAseq studies and comprises experiment performed in triplicate. The aim of this study was to examine the influence of C6orf203 silencing on mitochondrial transcriptome. To this end we engineered two stable cell lines with the use of human 293 Flp-In T-Rex cells as parental. First cell line inducible expressed miRNAs silencing endogenous copy of C6orf203 gene while second one expressed additionally transgenic version of FLAG-tagged C6orf203 which contained silent mutations causing insensitivity to miRNA. We also analyzed RNA isolated from parental 293 Flp-In T-Rex cells. RNAseq libraries were prepared with the use of strand-specific library preparation procedures. RNAs were random fragmented and reverse transcribed using random oligomers as primers (dUTP-based protocol, see PMID: 29590189, PMID: 22609201; this pipeline enables analysis of RNAs (> ~100 nucleotides)). RNA was isolated from unfractionated cells using TRI-Reagent. Before preparation of the libraries total RNA was subjected to depletion of nuclear-encoded rRNAs (Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat), Epicenter). Libraries were sequenced with the help of Illumina sequencing platform.

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Kotrys AV, Cysewski D, Czarnomska SD, Pietras Z, Borowski LS, Dziembowski A, Szczesny RJ