Modeling the nascent RNA transcriptome with chrTT-seq to extract chromatin dissociation dynamics on newly transcribed RNA.

To capture chromatin dissociation dynamics of nascent RNA transcripts we labeled MCF-7 cells with 4-thio-uridine (4-SU) for 8 minutes, then chased with an excess of uridine for 5, 10, 15 and 20 min. At each pulse-chase time point we performed TT-seq from the chromatin-associated and nucleoplasmic fraction. The ratios of spike-in normalized chromatin-associated to chromatin-released nascent RNA read coverage over genomic features (transcripts' last exons) were fitted on an exponential decay function to extract an estimated chromatin association half-live time for each transcript. Briefly, chromatin fractionation was performed at 0.5 M urea. RNA was extracted with acidic phenol from the chromatin-associated and chromatin-released fraction, and chemically fragmented (0.1 M NaOH on ice) before 4-SU based purification of newly transcribed RNA. A mix of three 4SU-labeled and three unlabeled ERCC spike-ins were added to RNA prior to fragmentation. Libraries for Illumina sequencing were constructed according to the TrueSeq Stranded mRNA Library Preparation Kit.

为捕获新生RNA转录本的染色质解离动力学，我们将MCF-7细胞以4-硫尿苷（4-thio-uridine，4-SU）标记8分钟，随后以过量尿苷进行脉冲追踪，追踪时长分别为5、10、15和20分钟。在每个脉冲追踪时间点，我们分别对染色质结合组分与核质组分开展TT-seq测序。将经外源spike-in标准化后的染色质结合与染色质游离新生RNA的读段覆盖度在基因组特征（转录本最后外显子）上的比值拟合至指数衰减函数，以提取每个转录本的染色质结合半衰期。简言之，染色质分离操作在0.5 M尿素条件下进行。使用酸性苯酚从染色质结合组分与染色质游离组分中提取RNA，并在基于4-SU纯化新生转录RNA之前，对RNA进行化学片段化（冰浴条件下以0.1 M NaOH处理）。在片段化前，向RNA样品中加入3种经4SU标记与3种未标记的ERCC外源spike-in对照混合物。按照TrueSeq Stranded mRNA Library Preparation Kit的操作流程构建Illumina测序文库。