Protocol for affordable and efficient profiling of nascent RNAs in bread wheat using GRO-seq

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Global Run-On sequencing (GRO-seq) is crucial for detecting transcriptional activity.
• High sequencing costs hinder the widespread adoption of GRO-seq.
• Existing GRO-seq protocols face challenges in data yield and affordability.

Purpose of the Study:

• To develop a more efficient and affordable GRO-seq protocol.
• To improve the yield of valid sequencing data.
• To enable detailed profiling of transcriptional activity, including enhancer transcription.

Main Methods:

• Incorporated a ribosomal RNA (rRNA) removal step into the GRO-seq protocol.
• Performed rRNA removal after nuclear RNA isolation and before nascent RNA immunoprecipitation.
• Applied the modified protocol to profile enhancer transcription in allohexaploid bread wheat.

Main Results:

• The modified GRO-seq protocol significantly reduces costs and increases efficiency.
• Achieved a 20-fold increase in the proportion of valid sequencing data.
• Successfully profiled enhancer transcription in allohexaploid bread wheat.

Conclusions:

• The optimized GRO-seq protocol overcomes cost barriers for widespread application.
• This method provides a powerful tool for studying transcriptional regulation in complex organisms.
• Enhanced data quality facilitates deeper insights into gene expression and regulatory elements.