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. Overall design: 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.