Overview: Capture the Dynamics of Transcription Beyond Steady-State
Standard RNA sequencing (RNA-seq) is a powerful tool, but it has a fundamental blind spot. It only measures the "steady-state" level of RNA—the total accumulated inventory in the cell at a specific moment. It is analogous to taking a single snapshot of a busy highway: you can count the cars, but you cannot tell how fast they are moving, where traffic is jamming, or which cars just entered the road.
Transient Transcriptome Sequencing (TT-Seq) transforms that snapshot into a movie. By metabolically labeling newly transcribed RNA for a very short duration (typically 5–10 minutes), TT-Seq allows researchers to measure the "fourth dimension" of the transcriptome: time. This method quantifies exactly how fast RNA is being produced (synthesis rate) and how fast it is being broken down (degradation rate) with single-nucleotide resolution.
CD Genomics offers a comprehensive, end-to-end TT-Seq service designed to decode these hidden kinetics. We enable you to move beyond simple differential expression to understand the precise regulatory mechanisms driving gene changes. This approach is essential for dissecting rapid biological responses, such as drug mechanism-of-action, acute immune activation, or immediate-early gene induction. Our service is "Research Use Only" (RUO) and is optimized for high sensitivity, ensuring the detection of unstable molecules that standard methods miss, such as enhancer RNAs (eRNAs) and upstream antisense RNAs (uaRNAs).
The Power of Kinetic Profiling
While standard RNA-seq measures abundance (the result of production minus decay), TT-Seq measures the rates directly. This distinction provides three critical advantages:
- Real-Time Responsiveness: Detect immediate changes in transcription initiation minutes after a stimulus, long before total RNA levels reflect the change.
- Unparalleled Sensitivity: Capture short-lived transcripts (like eRNAs) that are degraded so rapidly they never accumulate enough to be detected by standard Poly-A or Ribodepletion protocols.
- Decoupling Regulation: Distinguish whether a gene's downregulation is caused by a block in synthesis (transcriptional inhibition) or an increase in decay (post-transcriptional destabilization).
Principles of Technology: How TT-Seq Works
To appreciate the superiority of TT-Seq for kinetic studies, it is important to understand the underlying chemistry and how it differentiates nascent RNA from the stable pool.
Step 1: Metabolic Labeling with 4sU
The process begins with the addition of 4-thiouridine (4sU) to the cell culture medium. Living cells actively uptake 4sU and incorporate it into nascent RNA chains. By limiting this "pulse" to a short window (e.g., 5 minutes), we ensure that only the RNA synthesized during this specific timeframe carries the 4sU label.
Step 2: Stopping the Reaction
Precision is key. We instantly halt the labeling process using Trizol or a similar lysis buffer to "freeze" the transcriptional state. The sample now contains pre-existing (unlabeled) RNA and nascent (thiol-labeled) RNA.
Step 3: Fragmentation (The Key Difference)
This is the critical differentiator between TT-Seq and other methods like standard 4sU-seq or SLAM-seq. Before separation, we fragment the total RNA. This reduces steric hindrance and ensures that a labeled nucleotide at the start of a 10kb transcript allows the entire transcript to be represented.
Step 4: Biotinylation and Enrichment
We treat the fragmented RNA with HPDP-biotin, which forms a disulfide bond with the 4sU thiol groups. Streptavidin-coated magnetic beads then capture the labeled RNA with high affinity (Kd ≈ 10-14). Stringent washing removes unlabeled background, and a reducing agent elutes the pure, nascent RNA.
Step 5: Sequencing and Analysis
The eluted nascent RNA allows us to construct a library representing the "active" transcriptome. By integrating this data with the labeling time and spike-in controls, we model the kinetics of every gene.
