Dataset Information

Mapping and quantifying nascent transcript start sites using TT-TSS-seq

ABSTRACT: Transcription initiation is a highly dynamic and tightly regulated process involving the coordinated action of transcription factors, chromatin remodelers, and RNA polymerase which determine where and when transcription begins. Accurately mapping and quantifying transcription start sites (TSSs) from nascently transcribed RNAs remains a key area of interest, as it provides critical insights into transcription dynamics. Here, we combined transient transcriptome sequencing with transcription start site sequencing (TT-TSS-seq) to accurately map and quantify transcription initiation sites from nascent transcripts. Since transient metabolic labelling yields low-input RNA, we optimized the TSS-seq protocol to enhance sensitivity and accuracy. Specifically, we refined enzymatic reactions for decapping and RNA ligation and incorporated 5' oligonucleotides including unique molecular identifiers (UMIs) and barcodes to enable accurate quantification and sample multiplexing. The TT-TSS-seq approach detected transcription initiation of unstable transcripts, such as enhancer RNAs. Moreover, we identified that a large fraction of genes use multiple transcription initiation sites, yet often produce only a single stable transcript. Overall, TT-TSS-seq provides precise mapping and quantification of transcription initiation sites, offering new insights into transcriptional dynamics and expanding the toolkit for studying gene regulation.

ORGANISM(S): Mus musculus Saccharomyces cerevisiae

PROVIDER: GSE292786 | GEO | 2025/03/31

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Background Bacteria rely on efficient gene regulatory mechanisms to switch between genetic programs when they are facing new environments. Although this regulation can occur at many different levels, one of the key steps is the initiation of transcription. Identification of the first nucleotide transcribed by the RNA polymerase is therefore essential to understand the underlying regulatory processes, since this provides insight on promoter strength and binding sites for transcriptional regulators, and additionally reveals the exact 5' untranslated region of the transcripts, which often contains elements that regulate translation. Results Here we present data from a novel TSS-EMOTE assay (Transcription Start Specific Exact Mapping Of Transcriptome Ends) to precisely map the transcription initiation sites of four entire transcriptomes. TSS-EMOTE is a variation of the dRNA-seq method, which has been combined with the EMOTE protocol, in order to increase detection of longer transcripts and limit biases introduced by PCR amplification of the Illumina sequencing library. Using TSS-EMOTE, 2018 promoters were detected in the opportunistic pathogen Staphylococcus aureus, and detailed consensus sequences could be obtained for the RNA polymerase recognition elements (e.g. sigma factor binding sites). The data also revealed a 94 nt median length of the 5' untranslated region in S. aureus, giving important insights for future work on translational regulation. Additionally, the transcriptomes of three other opportunistic pathogens, Staphylococcus epidermidis, Acinetobacter baumannii and Enterobacter aerogenes, were examined, and the identified promoter locations were then used to generate a map of the operon structure for each of the four organisms. Conclusions Mapping transcription start sites, and subsequent correlation with the genomic sequence, provides a multitude of important information about the regulation of gene expression, both at the transcriptional and translational level, by defining 5' untranslated regions and sigma-factor binding sites. We have here mapped transcription start sites in four important pathogens using TSS-EMOTE, a method that works with both long and 3'-phosphorylated RNA molecules, and which incorporates Unique Molecular Identifiers (UMIs) to allow unbiased quantification.

Dataset Information

Mapping and quantifying nascent transcript start sites using TT-TSS-seq

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets