biostudies-literatureUnknown37(9-10)Yu ADA wide range of sequencing methods has been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed Butt-seq (bulk analysis of nascent transcript termini sequencing), which can produce libraries from purified nascent RNA in 6 h and from as few as 10,000 cells-an improvement of at least 10-fold over existing techniques. Butt-seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcriptiGenes & development432-448https://www.ebi.ac.uk/biostudies/studies/S-EPMC10270195biostudies-literatureButt-seq: a new method for facile profiling of transcription.PMC10270195Rosbash MYu ADfalseButt-seq: a new method for facile profiling of transcription.A wide range of sequencing methods has been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed Butt-seq (bulk analysis of nascent transcript termini sequencing), which can produce libraries from purified nascent RNA in 6 h and from as few as 10,000 cells-an improvement of at least 10-fold over existing techniques. Butt-seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcripti2023-01-01T00:00:00Z2023 May2026-05-28T12:10:28.24Z2025-02-19T00:50:12.483ZS-EPMC102701953716464510.1101/gad.350434.123