Expression profiling by high throughput sequencing Other Genome binding/occupancy profiling by high throughput sequencing
Transcription is a highly dynamic process that generates the majority of single-stranded DNA in the genome as ‘transcription bubbles’. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq), which marks single-stranded DNA in situ for genome-wide capture and sequencing, based on the fast and specific reaction between N3-kethoxal and guanines in single-stranded DNA in live cells. KAS-seq enables rapid (within 5 min) and sensitive detection of the position, relative level, and dynamics of ssDNA, as a readout of transcriptionally engaged RNA polymerases, with 1,000 cells. KAS-seq defines a group of enhancers that are single-stranded, which confers higher transcription factor binding density, more enhancer-promotor interactions, and higher ability to activate transcription. Under protein condensation inhibition condition, KAS-seq reveals unprecedented, rapid release of RNA polymerase II (Pol II) from a group of promotors, and reveals gene sets with different extents of responses, which correlate with Pol II C-terminal domain (CTD) serine 5 phosphorylation (S5P) level at TSS under no treatment condition. KAS-seq thus facilitates fast, comprehensive, and accurate analysis of transcription dynamics and enhancer activities simultaneously in a low input and high-throughput manner.
Examination of N3-kethoxal labeling ssDNA pattern in 2 HEK293 and mESCs.
Examination of N3-kethoxal labeling ssDNA pattern in mouse liver tissue.