Using TTchem-seq for profiling nascent transcription and measuring transcript elongation
Using TTchem-seq for profiling nascent transcription and measuring transcript elongation
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DOI：
10.1038/s41596-019-0262-3
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发表时间：
2020-01-08
期刊：
影响因子：
14.8
通讯作者：
Svejstrup, Jesper Q.
中科院分区：
文献类型：
--
作者：
Gregersen, Lea H.;Mitter, Richard;Svejstrup, Jesper Q.
转录动力学可以通过对新生RNA和新合成RNA的高通量测序在全基因组范围内进行研究。4-硫脲（4SU）在体内标记可以特异性捕获这些新的转录本，4SU残基被生物素连接物标记，并在文库生产和高通量测序之前使用链亲和素珠捕获。为了获得转录区域的高分辨率剖面，在生物素标记之前引入了RNA片段化步骤，这种方法被称为瞬时转录组测序（TT-seq）。我们最近介绍了一种化学方法，我们称之为TTchem-seq。我们描述了TTchem-seq如何与可逆CDK9抑制剂5,6-二氯苯并咪唑1- β -d-核呋喃苷（DRB）的早期伸长瞬时抑制结合使用，以测量体内RNA聚合酶II （RNAPII）的伸长率，我们称之为DRB/TTchem-seq技术。在这里，我们提供了执行TTchem-seq和DRB/TTchem-seq的详细协议，包括计算分析。实验和数据分析可以在10-13天的时间内进行，需要分子生物学和生物信息学技能。4-硫脲（4SU）标记新生转录物可以对哺乳动物细胞中新转录的rna进行高分辨率全基因组分析。结合瞬时转录抑制，可以测量RNA聚合酶II的延伸速度。
The dynamics of transcription can be studied genome wide by high-throughput sequencing of nascent and newly synthesized RNA. 4-thiouridine (4SU) labeling in vivo enables the specific capture of such new transcripts, with 4SU residues being tagged by biotin linkers and captured using streptavidin beads before library production and high-throughput sequencing. To achieve high-resolution profiles of transcribed regions, an RNA fragmentation step before biotin tagging was introduced, in an approach known as transient transcriptome sequencing (TT-seq). We recently introduced a chemical approach for RNA fragmentation that we refer to as TTchem-seq. We describe how TTchem-seq can be used in combination with transient inhibition of early elongation using the reversible CDK9 inhibitor, 5,6-dichlorobenzimidazole 1-beta-d-ribofuranoside (DRB), to measure RNA polymerase II (RNAPII) elongation rates in vivo, a technique we call DRB/TTchem-seq. Here, we provide detailed protocols for carrying out TTchem-seq and DRB/TTchem-seq, including computational analysis. Experiments and data analysis can be performed over a period of 10-13 d and require molecular biology and bioinformatics skills.4-thiouridine (4SU) labeling of nascent transcripts enables high-resolution genome-wide profiling of newly transcribed RNAs in mammalian cells. In combination with transient transcription inhibition, RNA polymerase II elongation speeds can be measured.