Transient transcriptome sequencing: experimental protocol to monitor genome-wide RNA synthesis including enhancer transcription v1

Transcriptome analysis by RNA sequencing (RNA-seq) measures the steady-state abundance of cellular RNA, which is a result of the interplay of RNA synthesis and RNA degradation. In order to measure RNA synthesis, RNA can be labeled with 4-thiouridine (4sU) in cells, purified and sequenced (4sU-seq). Although 4sU-seq has a higher sensitivity than RNA-seq, it is not sensitive enough to reliably detect short-lived (transient) RNAs such as enhancer, antisense, and promoter-associated RNAs synthesized from large genomes such as the human genome. This is because when the 4sU labeling time is less than 30 min only a short 3’-region of transcripts is labeled, and a long pre-existing unlabeled 5’ region leads to a 5’-bias in the sequencing data. Transient transcriptome sequencing (TT-seq) overcomes this limitation by combining a short 4sU labeling pulse with an RNA fragmentation step. The labeled, newly synthesized RNA fragments are purified and sequenced, resulting in a very low fraction of contaminating non-labeled RNA. TT-seq is easy to use and includes RNA spike-in controls for global normalization between datasets from different samples. TT-seq enables studies of the kinetics of RNA metabolism, and mechanistic studies of transcription regulation. Also, TT-seq is ideally suited to monitor rapid changes in gene activity as well as the dynamics of enhancer landscapes during transcription responses.