Systematic Evaluation of Parameters in RNA Cytosine Methylation Analysis

Here we systematically analysis bisulfite-treated RNA sequencing data (RNA BS-seq) for the purpose of identifying m5C methylation data from mouse neural stem cells. We isolated mitochondrial fractions to better understand the m5C characteristics of RNAs found in these cellular compartments. Throughout the analysis, we identify key parameters to be used in RNA m5C methylation analysis, and identify downstream effects of artifact detection on RNA m5C levels. In addition, we utilized Unique Molecular Identifiers (UMIs) in conjunction with ERCC (External RNA Controls Consortium) as RNA controls to identify rates of m5C artifcats generated by RNA duplication and PCR amplification error rates. Using various preparation parameters, we identify ideal preparation methods for RNA BS-seq. Overall design: Analysis of RNA BS-seq data across various RNA BS-seq preparation methods of mitochondrial isolates. Please note that processed data file generated from multiple samples is linked to the corresponding rep1 sample -or- to the Series records.