GROseq

GRO-seq, short for Global Run-On sequencing, is a high-throughput method for profiling genome-wide transcription by sequencing nascent RNA. The technique begins with isolation of nuclei from cells or tissues, followed by a nuclear run-on in the presence of labeled nucleotides. Engaged RNA polymerases extend their transcripts briefly, incorporating the labeled nucleotides into nascent RNA. The newly synthesized, labeled RNA is then isolated, converted to cDNA, and subjected to next-generation sequencing. Because the reads derive from actively transcribing polymerases, GRO-seq reports transcriptional activity rather than steady-state RNA abundance and is strand-specific, enabling detection of sense and antisense transcription.

Applications and interpretation: GRO-seq provides a snapshot of transcriptional activity across the genome, allowing analysis of promoter-proximal pausing, the distribution of RNA polymerase II, and the identification of actively transcribed enhancers and enhancer RNAs. It is used to study transcriptional responses to stimuli, developmental processes, and disease states. Compared with conventional RNA-seq, GRO-seq is more sensitive to immediate changes in transcription and can reveal regulatory dynamics that are not captured by RNA abundance alone.

Limitations and related methods: GRO-seq generally requires substantial input material and high-quality nuclei, and it does not capture post-transcriptional processing events. Resolution is influenced by run-on duration and library preparation. Related approaches include PRO-seq (Precision Run-On sequencing), which can offer higher resolution, and GRO-cap, which maps transcription start sites. GRO-seq originated in the late 2000s, with foundational work by Core, Waterfall, and Lis, demonstrating widespread promoter-proximal pausing and nascent transcription in mammalian cells.