Systematic evaluation and optimization of cDNA library preparation for next-generation sequencing

Abstract

RNA G-quadruplex (rG4), the secondary structure which forms from stacks of G-quartets in the RNA consists of G-rich sequences, are found to regulate the essential cellular processes in organisms. Moreover, recent research found that rG4 regulates the genes that related to several diseases, including neurological disorder and cancers. Hence, understanding the biology of rG4-containing RNAs might be beneficial to analysis and therapeutic strategy development towards corresponding diseases. cDNA library preparation is important for many high-throughput sequencing applications, such as rG4-seq. A systematic evaluation on the procedures of the experimental pipeline, however, is lacking. Herein, we perform a comprehensive assessment of the 5 key experimental steps involved in the cDNA library preparation of rG4-seq, and identify better reaction conditions and/or enzymes to carry out each of these key steps. Notably, we benchmark on the RNA input requirement for this optimized experimental pipeline, and show that only sub-nanogram level of RNA is required for this new and sensitive approach. In addition, the time to perform these steps is substantially reduced to hours. Our method and results can be directly applied in protocols that require cDNA library preparation, and provide insights to the further development of simple and efficient cDNA library preparation for different biological applications.