rG4-seq 2.0: enhanced transcriptome-wide RNA G-quadruplex structure sequencing for low RNA input samples

RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles such as transcription termination, alternative splicing, mRNA localization and stabilization and translational process. We recently developed RNA G-quadruplex structure sequencing (rG4-seq) technique and discovered many rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from complicated gel purification step and limited PCR product yield and thus requires a high RNA input amount, limiting its applications for physiologically or clinically relevant studies. In this study, we have developed rG4-seq 2.0 by introducing a new 5' adapter containing deoxyuridine in the library preparation to enhance the library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produced high quality cDNA libraries that supported reliable and reproducible rG4 identification at varying RNA inputs (as low as 10 ng amount of RNA). rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. Our new method can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods.