Zhang Jie, Liang Zhixian, Sun Mingxin, Hebenstreit Daniel, Zhang Shaohui
National Engineering Research Center for Healthcare Devices & Guangdong Provincial Key Laboratory of Medical Electronic Instruments and Materials, Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China.
National Engineering Research Center for Healthcare Devices, Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China.
Bio Protoc. 2025 Jul 20;15(14):e5395. doi: 10.21769/BioProtoc.5395.
Transcriptional pausing dynamically regulates spatiotemporal gene expression during cellular differentiation, development, and environmental adaptation. Precise measurement of pausing duration, a critical parameter in transcriptional control, has been challenging due to limitations in resolution and confounding factors. We introduce Fast TV-PRO-seq, an optimized protocol built on time-variant precision run-on sequencing (TV-PRO-seq), which enables genome-wide, single-base resolution mapping of RNA polymerase II pausing times. Unlike standard PRO-seq, Fast TV-PRO-seq employs sarkosyl-free biotin-NTP run-on with time gradients and integrates on-bead enzymatic reactions to streamline workflows. Key improvements include (1) reducing experimental time from 4 to 2 days, (2) reducing cell input requirements, and (3) improved process efficiency and simplified command-line operations through the use of bash scripts. Key features • Reduces experimental duration from 4 to 2 days via on-bead enzymatic reactions and streamlined workflows. • Enables single-nucleotide resolution pausing time mapping using time-variant biotin-NTP run-on with saturation kinetics. • Compatible with reduced cell input (10-10 cells) and sarkosyl-free conditions for improved experimental feasibility. • Integrates bash scripts and simplified commands for enhanced reproducibility and reduced computational complexity.
转录暂停在细胞分化、发育和环境适应过程中动态调节基因表达的时空特性。转录控制中的一个关键参数——暂停持续时间的精确测量，由于分辨率的限制和混杂因素而一直具有挑战性。我们引入了Fast TV-PRO-seq，这是一种基于时变精确延伸测序（TV-PRO-seq）优化的方案，它能够对RNA聚合酶II的暂停时间进行全基因组、单碱基分辨率的图谱绘制。与标准PRO-seq不同，Fast TV-PRO-seq采用无十二烷基肌氨酸的生物素-NTP延伸并带有时间梯度，并整合了磁珠上的酶促反应以简化工作流程。主要改进包括：（1）将实验时间从4天缩短至2天，（2）降低细胞输入要求，以及（3）通过使用bash脚本提高了过程效率并简化了命令行操作。关键特性 • 通过磁珠上的酶促反应和简化的工作流程将实验持续时间从4天缩短至2天。 • 使用具有饱和动力学的时变生物素-NTP延伸实现单核苷酸分辨率的暂停时间图谱绘制。 • 与减少的细胞输入（10-10个细胞）和无十二烷基肌氨酸条件兼容，以提高实验可行性。 • 整合bash脚本和简化命令以提高可重复性并降低计算复杂性。
