Systematic evaluation and optimization on the experimental steps in RNA G-quadruplex structure sequencing

Multitudes of cDNA library preparation methods have been developed as a result of the recent exploration and investigation of the novel features of RNA, such as RNA structures. However, most of these methods require high RNA input (microgram of RNA), numerous processing and purification steps, and thus long cDNA library preparation time (few days). As such, a simple and efficient cDNA library preparation is critical to the further improvement of existing protocols, development of new transcriptome-wide methods and many other biochemical assays.Recently, we have developed RNA G-quadruplex structure sequencing (rG4-seq), and reported the prevalent in vitro formation of RNA G-quadruplex structures in the human transcriptome, providing a useful resource for further in vivo rG4 structural and functional characterization. The rG4-seq protocol requires high input of RNA (~500 ng RNA), and lengthy library preparation (~1.5 days). In addition, an extensive assessment on the procedures of the experimental pipeline of rG4-seq is currently lacking.In this study, we have systematically evaluated and optimized the 5 key experimental steps on the experimental pipeline of rG4-seq, and identified better reaction conditions and/or enzymes to carry out each of these key steps. We applied the improved methods to fragmented cellular RNA and generated two new cDNA libraries using reduced RNA inputs (~250 ng and ~50 ng RNA). The new libraries were found to have lower transcript abundance variations and lower 5’ and 3’ transcript coverage bias when compared to prior art. Moreover, the total time of the library preparation has been greatly reduced to several hours. Our method and results can be directly applied in protocols that require cDNA library preparation, and provided insights to the further development of simple and efficient cDNA library preparation for different biological applications.