Processing and evaluating the quality of genome-wide nascent transcription profiling libraries

Precision genomic run-on assays (PRO-seq) quantify nascent RNA at single nucleotide resolution with strand specificity. Here we deconstruct a recently published genomic nascent RNA processing pipeline (PEPPRO) into its components and link the analyses to the underlying molecular biology. PRO-seq experiments are evolving and variations can be found throughout the literature. The analyses are presented as individual code chunks with comprehensive details so that users can modify the framework to accommodate different protocols. We present the framework to quantify the following quality control metrics: library complexity, nascent RNA purity, nuclear run-on efficiency, alignment rate, sequencing depth, and RNA degradation.