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

• Published in: STAR protocols 2022-09, Vol.3 (3), p.101657-101657, Article 101657
• Main Authors: Chen, Yan, Zhu, Jiafu, Xie, Yilin, Li, Zijuan, Zhang, Yijing, Liu, Min, Dong, Zhicheng
• Format: Article
• Language: English
• Subjects: Bioinformatics; Molecular biology; Plant sciences; Protocol; RNAseq; Sequence analysis; Sequencing
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101657

Exorbitant sequencing cost is one of the main obstacles limiting the widespread application of Global Run-On sequencing (GRO-seq) to detect transcriptional activity. Here, we describe a more efficient and affordable protocol for GRO-seq that incorporates an rRNA removal step after nuclear RNA isolation and before nascent RNA immunoprecipitation. We have successfully applied this protocol to profile enhancer transcription in allohexaploid bread wheat and increased the proportion of valid data by 20 times. For complete details on the use and execution of this protocol, please refer to Xie et al. (2022). [Display omitted] •The detailed GRO-seq protocol integrates rRNA depletion•Cost-efficient GRO-seq protocol for detection of eRNA in bread wheat•Applicable for any large complex plant or animal genomes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Exorbitant sequencing cost is one of the main obstacles limiting the widespread application of Global Run-On sequencing (GRO-seq) to detect transcriptional activity. Here, we describe a more efficient and affordable protocol for GRO-seq that incorporates an rRNA removal step after nuclear RNA isolation and before nascent RNA immunoprecipitation. We have successfully applied this protocol to profile enhancer transcription in allohexaploid bread wheat and increased the proportion of valid data by 20 times.