Next generation sequencing (NGS) is routinely used in gene expression analyses. In particular, RNA-seq has been the method of choice for highly sensitive genome-wide quantification of RNA expression. The method can be used in a wide variety of model systems, including studies to gain insight into underlying mechanisms of toxicologic processes and disease development induced by environmental toxicants. RNA-seq has also been coupled to many other molecular biology protocols to monitor specific aspects of the gene expression process. Here, we describe two such coupling—(a) global run-on sequencing (GRO-seq) that coupled it to the nuclear run-on (NRO), and (b) polysome profiling that coupled it to sucrose-gradient-based polysome isolation. Simultaneous RNA-seq, GRO-seq, and polysome profiling analyses enabled genome-wide analysis of the mode of stability control of individual RNA molecules.