Amplicons of a ND5 mRNA segment to detect m1A1374 levels in HEK pTRMT10C cells and control cells
This experiment was set up to assess the effect of TRMT10C overexpression on m1A methylation levels at position 1374 of the mitochondrial encoded ND5 mRNA. To investigate if TRMT10C is the writer enzyme for this m1A site, a cell system providing tetracycline-inducible TRMT10C expression was used (termed pTRMT10C cells). To control the effect of tetracycline itself, a corresponding control cell line without the tetracycline-inducible TRMT10C plasmid was used (termed Ctl cells). Method description: pTRMT10C and Ctl cells were incubated with three different concentrations of tetracycline (0 µg/mL, 0.1 µg/mL and 1 µg/mL) for 24 hours. The RNA was isolated using a Trizol-based protocol. Afterwards a Reverse transcription was performed with the RT SuperScript-IV (SS-IV) and a ND5 specific RT primer. Next, PCR was conducted to amplify the sequence around the m1A position. The amplicon is about 100 bp of length and was sequenced in an Illumina Sequencing MiSeq 2x75 PE run. For both cell lines the m1A analysis method was conducted with 4 biological replicates for each of the three tetracycline concenctraion.