What Is GRO-seq and How Does It Measure Transcription?

Global Run-On Sequencing (GRO-seq) is a method that provides a snapshot of gene activity within a cell at a specific moment. It is designed to measure the rate of transcription, the process of copying genetic information from DNA into RNA. By capturing only the RNA molecules being synthesized, GRO-seq identifies which genes are actively being transcribed, giving a dynamic picture of the genome’s transcriptional output.

The GRO-seq Experimental Process

The GRO-seq procedure begins by isolating intact nuclei from cells, which effectively halts the natural transcription process. This preserves the RNA polymerase enzymes at the exact locations on the DNA where they were actively synthesizing RNA. This “freezing” of activity is what allows for a moment-in-time measurement of gene expression.

Following nuclear isolation, the “run-on” reaction is initiated. Researchers add a solution to the nuclei containing labeled RNA building blocks, such as 5-Bromouridine 5′-triphosphate (Br-UTP). The RNA polymerase enzymes that were paused on the DNA resume transcription briefly, incorporating these labeled molecules into the growing RNA strands. This step ensures that only the RNA actively being made at the moment of nuclear isolation becomes labeled.

The next phase involves isolating only the newly synthesized and labeled RNA. This is achieved using antibodies that specifically recognize and bind to the bromine tag (Br-UTP) incorporated into the RNA. These antibodies are attached to microscopic beads, which allows for the selective capture of the nascent RNA molecules, separating them from all other RNA present in the nucleus.

Once the labeled nascent RNA is purified, it is prepared for high-throughput sequencing. The RNA is broken down into smaller fragments, and enzymes are used to convert these RNA fragments into a more stable form called complementary DNA (cDNA). This cDNA library is then loaded into a sequencing machine, which reads the sequence of each fragment.

Data Generated from GRO-seq

The raw output from a GRO-seq experiment is a large collection of short DNA sequences. These sequences are mapped back to the organism’s reference genome to determine their precise location. The presence of sequencing reads aligned to a particular gene indicates it was being transcribed, allowing scientists to create a genome-wide map of all active genes.

The density of the sequencing reads provides a quantitative measure of transcription. A higher number of reads mapping to a specific gene indicates a higher rate of transcription. This allows researchers to determine the relative activity of different genes within the same cell or compare the transcriptional activity of a single gene under different conditions.

GRO-seq can also identify and map the precise location of regulatory elements. It pinpoints Transcription Start Sites (TSSs), where transcription begins, and detects transcription at enhancers, which are DNA sequences that regulate gene activity from a distance. GRO-seq captures the short, unstable RNA molecules produced from these enhancers, known as enhancer RNAs (eRNAs), indicating that the enhancer is active.

Key Research Applications

GRO-seq is well-suited for studying dynamic cellular responses to stimuli. Researchers use it to track rapid changes in gene transcription that occur when cells are exposed to hormones, drugs, or environmental stresses. By capturing the immediate transcriptional output, scientists can observe the first wave of gene activation or repression that orchestrates the cell’s response, often within minutes.

The technique is also instrumental in studies of gene regulation. It helps identify the direct targets of specific transcription factors—the proteins that control which genes are turned on or off. By depleting a transcription factor and using GRO-seq to see which genes show altered transcription rates, researchers can map out complex gene regulatory networks.

Another application of GRO-seq is distinguishing between different modes of gene regulation. Gene expression can be controlled at the level of transcription or at later stages, such as by altering RNA stability (post-transcriptional regulation). Because GRO-seq directly measures the synthesis of new RNA, it allows scientists to isolate and study transcriptional regulation specifically.

Distinctions from Other Transcriptomic Methods

A primary distinction exists between GRO-seq and the more common RNA-seq. GRO-seq measures nascent RNA, providing a real-time measurement of the rate of transcription. In contrast, standard RNA-seq measures the total amount of mature RNA in a cell, which represents a steady-state level that is the net result of both RNA synthesis and degradation. The difference can be visualized by imagining a bucket: GRO-seq measures the rate water flows from the tap, while RNA-seq measures the total water in the bucket.

This focus on nascent transcripts allows GRO-seq to detect very unstable RNA molecules that are often missed by RNA-seq. Many regulatory RNAs, such as eRNAs, are transcribed but then rapidly degraded. Because RNA-seq relies on the accumulation of RNA, these transient molecules are often invisible in the data. GRO-seq captures them as they are being made, providing a more complete picture of the transcriptional landscape.

A related technique, Precision Run-On sequencing (PRO-seq), is a refinement of GRO-seq. While based on the same principles, PRO-seq offers a higher resolution, capable of mapping the exact position of the RNA polymerase enzyme to a single nucleotide. This precision allows for a more detailed view of the transcription process, such as identifying the precise locations where polymerases pause.