A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq)

Tracking active transcription with the nuclear run-on (NRO) assays has been instrumental in uncovering mechanisms of gene regulation. The coupling of NROs with high-throughput sequencing has facilitated the discovery of previously unannotated or undetectable RNA classes genome-wide. Precision run-on sequencing (PRO-seq) is a run-on variant that maps polymerase active sites with nucleotide or near-nucleotide resolution. One main drawback to this and many other nascent RNA detection methods is the somewhat intimidating multi-day workflow associated with creating the libraries suitable for high-throughput sequencing. Here, we present an improved PRO-seq protocol where many of the enzymatic steps are carried out while the NRO RNA remains bound to magnetic beads. These adaptations reduce time, and we demonstrate that the resulting libraries are of the same quality as libraries generated using the original published protocol. The faster library preparation procedure decreases opportunity for sample loss and RNA degradation. Overall design: Two replicate libraries of PRO-seq were generated using 1M K562 cells as input material with our new, quick PRO-seq protocol (qPRO-seq). As a comparison, we also generated two replicate libraries with identical input material (1M K562 cells) using published protocol (PMID: 27442863). We also demonstrated our new protocol is robust to low-input samples by generating two replicate libraries with the qPRO-seq protocol using 250k K562 cells as input.