Next Generation Sequencing-based analysis of RNA polymerase functions

Next Generation Sequencing (NGS) that revolutionized genome wide studies allows analysis of complex nucleic acids mixtures containing thousands of sequences. This extraordinary analytical power of NGS can be harnessed for the analysis of in vitro experiments where DNA template sequence dependence of protein activity acting on DNA can be studied in a single reaction for thousands of DNA sequence variants. This allows a rapid accumulation of data on DNA sequence dependence of the process of interest to a depth not accessible by standard experimentation. We use an example of bacterial RNA polymerase promoter melting activity to describe the NGS-based methodology to study DNA template dependence of protein activity.