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 TT_chem-seq. We describe how TT_chem-seq can be used in combination with transient inhibition of early elongation using the reversible CDK9 inhibitor, 5,6-dichlorobenzimidazole 1-β-d-ribofuranoside (DRB), to measure RNA polymerase II (RNAPII) elongation rates in vivo, a technique we call DRB/TT_chem-seq. Here, we provide detailed protocols for carrying out TT_chem-seq and DRB/TT_chem-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.

通过对新生和新合成的 RNA 进行高通量测序，可以在全基因组范围内研究转录动力学。体内 4-硫尿苷 (4SU) 标记能够特异性捕获此类新转录物，其中 4SU 残基由生物素接头标记，并在文库生成和高通量测序之前使用链霉亲和素珠捕获。为了获得转录区域的高分辨率图谱，在生物素标记之前引入了 RNA 片段化步骤，这种方法称为瞬时转录组测序 (TT-seq)。我们最近介绍了一种用于 RNA 片段化的化学方法，我们称之为 TT _chem -seq。我们描述了 TT _chem如何-seq 可与使用可逆 CDK9 抑制剂 5,6-二氯苯并咪唑 1-β- d-呋喃核糖苷 (DRB) 对早期延伸的瞬时抑制结合使用，以测量体内 RNA 聚合酶 II (RNAPII) 延伸率，这是一种技术我们称之为 DRB/TT _chem -seq。在这里，我们提供了执行 TT _chem -seq 和 DRB/TT _chem -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，并且需要分子生物学和生物信息学技能。