Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0

Abstract

Background RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue. Results We developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance. Conclusions rG4-seq 2.0 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.

The multivalent G-quadruplex (G4)-ligands MultiTASQs allow for versatile click chemistry-based investigations

“…, ligands) in a quantitative manner. The modularity of these protocols, which are implementable in either a targeted (RT-qPCR analysis) or genome-wide manner (sequencing), makes them fully complementary to existing methods including G4-seq 46 using the G4 ligand PDS, 14 or G4 ChIP-seq, 47 BG4-ChIP-seq, 48 G4P ChIP-seq 49 and G4 CUT&Tag 50,51 using the G4-specific antibody BG4 52 (CUT&Tag was also developed with the nanobody SG4) 53 for DNA G4s, and rG4-seq 54,55 using PDS, or BG4-RNA-IP 56 and rG4IP 57 using BG4 for RNA G4s, which altogether constitute a unique array of techniques to accurately portray G4 biology. Second, the in situ click imaging of G4s provides a more qualitative but also straightforward way to track G4s in cells.…”

Regulation of Plant Translation under Abiotic Stress. A Molecular Orchestration

“…Modulation of the GQ structure is based on the selectivity of this motif for potassium ions, i.e., in the presence of K + a G-quadruplex is formed, but not in the presence of Li + [159]. This approach has been further developed and improved for full transcriptome search and GQS analysis [160,161]. Along with this, there is a method based on the precipitation of RNA containing GQS and further sequencing of these molecules [162].…”

A Molecular Orchestration of Plant Translation under Abiotic Stress

“…The modulation of the GQ structure is based on the selectivity of this motif for potassium ions Figure 4, i.e., a G-quadruplex is formed in the presence of K + but not in the presence of Li + [115]. This approach has been further developed and improved for full transcriptome searches and GQS analysis [116,117]. There is also a method based on the precipitation of RNA containing GQS and further sequencing of these molecules [118].…”