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-β-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.