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

Area of Science:

• Molecular Biology
• Genomics
• RNA Structure

Background:

• RNA G-quadruplexes (rG4s) are crucial regulatory motifs in various cellular processes.
• The original rG4-seq technique was limited by complex purification and low PCR yield, requiring substantial RNA input.
• This limitation hindered studies using scarce biological samples.

Purpose of the Study:

• To redesign and enhance the rG4-seq workflow for improved efficiency and broader applicability.
• To overcome the limitations of high RNA input requirements in the previous rG4-seq method.
• To enable reliable rG4 identification in samples with limited RNA availability.

Main Methods:

• Developed rG4-seq 2.0, incorporating a deoxyuridine-containing ssDNA adapter.
• Streamlined library preparation by eliminating the gel purification step.
• Reduced PCR amplification cycles and increased PCR product yield.

Main Results:

• rG4-seq 2.0 generates high-quality cDNA libraries suitable for reproducible rG4 identification.
• The method reliably detects rG4s even with RNA inputs as low as 10 ng.
• rG4-seq 2.0 improved rG4 calling accuracy and reduced nucleotide bias compared to the previous version.

Conclusions:

• rG4-seq 2.0 significantly enhances the identification and study of rG4s in low-abundance transcripts.
• The optimized workflow provides valuable insights for improving cDNA library preparation in related sequencing methods.
• This advancement facilitates rG4 research in clinically relevant scenarios with limited RNA.