Rates of in situ transcription and splicing in large human genes. TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding. Osmium-mediated transformation of 4-thiouridine to cytidine as key to study RNA dynamics by sequencing. Defining the status of RNA polymerase at promoters. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Genome-wide mapping of yeast RNA polymerase II termination. Strand-specific, high-resolution mapping of modified RNA polymerase II. Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Purification of P-TEFb, a transcription factor required for the transition into productive elongation. 5,6-dichloro-1-beta- d-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro. Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq. RECQL5 controls transcript elongation and suppresses genome instability associated with transcription stress. Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay. TT-seq captures enhancer landscapes immediately after T-cell stimulation. Enriching s 4 U-RNA using methane thiosulfonate (MTS) chemistry. Solid phase chemistry to covalently and reversibly capture thiolated RNA. SCAF4 and SCAF8, mRNA anti-terminator proteins. Global quantification of mammalian gene expression control. TT-seq maps the human transient transcriptome. High-resolution sequencing and modeling identifies distinct dynamic RNA regulatory strategies. Thiol-linked alkylation of RNA to assess expression dynamics. MOV10 is a 5ʹ to 3ʹ RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3ʹ UTRs. 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells. Tracking distinct RNA populations using efficient and reversible covalent chemistry. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. Ultrashort and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Experiments and data analysis can be performed over a period of 10–13 d and require molecular biology and bioinformatics skills. Here, we provide detailed protocols for carrying out TT chem-seq and DRB/TT chem-seq, including computational analysis. 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. We recently introduced a chemical approach for RNA fragmentation that we refer to as TT chem-seq. 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). 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. The dynamics of transcription can be studied genome wide by high-throughput sequencing of nascent and newly synthesized RNA.
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