Most of our knowledge of transcriptional regulation is based on the study of mRNA abundance. The development of high-throughput approaches has revealed pervasive transcription in all genomes that have been investigated so far. This has uncovered a highly interleaved transcriptome organization that involves thousands of coding and non-coding RNAs. Our ability to study transcriptional regulation is further complicated by the complexity within each locus. Each gene expresses diverse transcript isoforms using alternative promoters, exons and terminators. This generates alternative RNA molecules, i.e. isoforms with different length and sequence from the same gene. Our lab has developed multiple approaches to investigate transcript isoform variation at the genome-wide level (TIF-Seq, TIF-Seq2…). We are further developing approaches to better characterize previously undistinguishable transcripts (e.g., those that arise from neighboring genes, short abortive transcripts...). We are currently expanding those approaches to simplify the HT characterization of Transcription start sites, polyadenylation sites and poly(A) length across multiple organims.