RT Journal
A1 Elgood Hunt, Eleanor
A1 Vivori, Claudia
A1 Mitter, Richard
A1 Agnadottir, Vedis
A1 van Werven, Folkert J.
T1 Mapping and quantifying nascent transcript start sites using TT-TSS-seq
JF Genome Research 
JO Genome Research 
YR 2026 
FD March 01 
VO 36 
IS 3 
SP 600 
OP 610 
DO 10.1101/gr.280726.125 
UL http://genome.cshlp.org/content/36/3/600.abstract 
AB 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 combine transient transcriptome sequencing with transcription start site sequencing (TT-TSS-seq) to accurately map and quantify transcription initiation sites from nascent transcripts. Because transient metabolic labeling yields low-input RNA, we optimize the TSS-seq protocol to enhance sensitivity and accuracy. Specifically, we refine enzymatic reactions for decapping and RNA ligation and incorporate 5′ oligonucleotides containing unique molecular identifiers (UMIs) and barcodes to enable accurate quantification and sample multiplexing. The TT-TSS-seq approach detects transcription initiation of unstable transcripts, such as enhancer RNAs. Moreover, we show 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.