A guide to Dynamic Transcriptome Analysis (DTA)

Total RNA levels in a cell are the consequence of two opposing mechanisms, namely RNA synthesis and RNA degradation. DTA allows monitoring these contributions in a non-perturbing manner. It is provided with a kinetic modeling approach capable of the precise determination of synthesisand decay rates, which is implemented in this package (see supplementary methods in [6]). DTA can be applied to reveal rate changes for all kinds of perturbations, e.g. in knock-out or point mutation strains, as responses to stress stimuli or in small molecule interfering assays like treatments through miRNA or siRNA inhibitors. The experimental setup for DTA requires culturing cells in the presence of a labeling substrate (e.g. 4 thiouridine (4sU) or 4 thiouracil (4tU)) for a certain amount of time. Until the extraction of the RNA samples, the analogous (labeling) substrate will be incorporated into newly transcribed RNA. This setup yields three types of RNA fractions: total cellular RNA, newly transcribed labeled RNA and pre-existing unlabeled RNA. All three fractions can subsequently be quantified through gene expression profiling on microarrays or next generation sequencing (RNAseq). The DTA package implements methods to process a given DTA experiment (total (T), labeled (L) and unlabeled (U) RNA measurements) and derive RNA synthesis and decay rate estimates. The package DTA is broadly applicable to virtually every organism. This manual is a hands-on tutorial and describes all functions and R commands that are required. The method is applied to real and synthetic data. Important quality control plots and error assessment to prove the reliability of the data and the estimation are discussed throughout this manual. For details on the theoretical background and a thorough description of the experimental design, we refer the reader to [6] (supplementary methods).