Amplicons of a ND5 mRNA segment to detect m1A1374 levels in HEK pTRMT10C cells and control cells. 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.

本实验旨在评估TRMT10C过表达对线粒体编码ND5 mRNA第1374位m1A（N1-甲基腺嘌呤）甲基化水平的影响。为探究TRMT10C是否为该m1A位点的写入酶，本研究使用了四环素诱导型TRMT10C表达细胞系统（命名为pTRMT10C细胞）。为控制四环素本身的干扰效应，同时设置了不含四环素诱导型TRMT10C质粒的对照细胞系（命名为Ctl细胞）。
实验方法如下：将pTRMT10C与Ctl细胞分别用三种浓度的四环素（0 µg/mL、0.1 µg/mL、1 µg/mL）孵育24小时。采用基于Trizol的提取方案分离总RNA，随后使用RT SuperScript-IV（SS-IV）反转录酶及ND5特异性反转录引物完成反转录反应。接下来通过PCR扩增该m1A位点上下游序列，扩增片段长度约为100 bp，随后采用Illumina MiSeq测序平台进行2×75 PE双端测序。针对两种细胞系的三种四环素浓度组别，本研究均设置了4个生物学重复，并统一采用m1A分析方法开展检测。