Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq
Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq
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DOI：
10.1038/nprot.2015.035
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发表时间：
2015-04-01
期刊：
影响因子：
14.8
通讯作者：
Oren, Moshe
中科院分区：
文献类型：
--
作者：
Fuchs, Gilad;Voichek, Yoav;Oren, Moshe
4sUDRB-seq在基因组规模上分别测量转录延伸速度和RNA聚合酶II（Pol II）转变为活性延伸（TAE）的速率对总体mRNA产生速率的不同贡献。它使用5，6-二氯-1-β-d-呋喃核糖基苯并咪唑（DRB）可逆抑制转录延伸，结合4-硫代尿苷（4sU）脉冲，标记新转录的RNA。去除DRB后，在几个时间点收集细胞，将标记的RNA生物素化，捕获在链霉亲和素珠上并测序。4sUDRB-seq能够比较不同发育阶段或不同细胞类型之间的延伸速度，并且它允许研究特定转录因子对转录延伸速度与TAE的影响。RNA制备需要大约4天才能完成，深度测序需要额外的大约4-11天加上1-3天用于生物信息学分析。实验方案需要基本的分子生物学技能，而数据分析需要生物信息学知识，特别是MATLAB和Linux环境。
4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes similar to 4 d to complete, with deep sequencing requiring an additional similar to 4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.