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High-Throughput Kinetic RNA Sequencing

SLAMseq Anabolic Kinetic RNA-Seq

SLAMseq Anabolic RNA-Seq labels newly synthesized (nascent) transcripts in cell culture experiments and detects an increasing signal of nascent RNA as they are being synthesized.

Accurate Time-resolved Analysis of RNA Synthesis

Tracking nascent RNA over time allows to determine RNA synthesis rates and delivers accurate time-resolved information of RNA synthesis in response to a stimulus. SLAMseq allows to demonstrate treatment effects with higher resolution compared to conventional methods. SLAMseq Anabolic RNA-Seq is applicable for research purposes, such as gene regulation studies or deciphering cell signaling pathways, or during drug discovery and development processes, such as target identification and validation or biomarker discovery.


SLAMseq delivers precise measurements for global transcript biosynthesis rates in living cells and allows to distinguish primary and secondary effects of treatments.

The SLAMseq Anabolic Kinetics Kit differentiates between nascent and existing RNA. At t0, modified nucleotides (S4U) are added to cell culture media, which results in labeling of newly synthesized RNA. Existing RNA remains unlabeled. At tx, the RNA synthesis is stopped by cell lysis and RNA isolation. Sampling at different intervals, tx, allows for measurement of transcript synthesis rates (Fig. 1). Specific measurement of nascent RNA levels provides insights into transcriptome-wide RNA synthesis dynamics.

Figure 1 | Anabolic kinetics labeling experiment time course. Culturing cells with S4U-containing media (extra­cellular [S4U], solid green line) changes the intracellular S4U concentration (dashed green line). Nascent RNA will be labeled starting from t0. Time course measurements determine RNA synthesis rates. Transcripts with fast (black solid line) and slow (gray solid line) synthesis rates can be distinguished by relative differences in the increase in S4U detection over time. S4U levels for individual transcripts are measured by counting sequencing reads with T > C conversions.

Distinguish newly synthesized (nascent) RNA from total RNA using SLAMseq Anabolic Kinetic 3’ mRNA-Seq with QuantSeq FWD V2 for cost-efficient RNA-Seq analysis of RNA synthesis, or the SLAMseq Kinetics Kit – Anabolic Kinetics Module paired with other library prep kits. Profile nascent RNA levels at specified time points for differential expression analyses and perform time course S4U labeling experiments to map RNA synthesis dynamics for individual transcripts.

Figure 2 | Read coverage of the Sox2 3’ UTR from a SLAMseq experiment. Nascent RNA (green) is labeled and decreases over time, despite stable total RNA levels (blue).

SLAMseq resolves transcript expression dynamics on a transcriptome-wide scale. Individual transcript RNA synthesis rates can be measured directly (Fig. 3). RNA synthesis rates are depending on gene regulation processes, such as blockers, enhancer, cofactors, and are transcript specific.

Figure 3 | S4U labeling kinetics experiments reveal individual RNA synthesis rates of four gene loci.

Combining SLAMseq with protein modulators, or drug treatments distinguishes direct (primary) and indirect (secondary) target responses (Fig. 4). Use SLAMseq to dissect signaling pathways underlying biological processes and characterize drug-target responses on the transcriptional level (Muhar, M et al., 2018).

Figure 4 | S4U labeling followed by drug treatment and early-stage sampling (time points: t1 – t2) measures mRNA stability changes to map sequential drug responses.

Find more information about SLAMseq data analysis here or contact us at


SLAMseq Anabolic Kinetic RNA-Seq allows to detect RNA synthesis rates over time in cell culture experiments in response to a stimulus, e.g., a fast-acting compound or drug candidate. Optimal concentration of labeling reagent must be determined during exploring phase to limit cytotoxicity.

Total time for Step:
Hands-on time for Step :
Cultured cells are treated for >30 min with fast-acting compound or drug. The incubation duration depends on the compound and cell type. Negative controls need to be applied.

Cultured cells are grown in fresh media with 4-Thiouridine (S4U) to allow S4U nucleotides to be incorporated into newly synthesized. Optimal concentration of S4U must be determined during exploring phase to avoid cell toxicity.

Cells are sampled at a given timepoint(s) and RNA is extracted under reducing conditions. The isolated total RNA contains both existing (labeled) and newly synthesized (unlabeled) RNA for catabolic kinetics experiments.

Total RNA (1 - 5 µg) is mixed with iodoacetamide (IAA), which modifies the 4-thiol group of S4U-containing nucleotides via the addition of a carboxyamidomethyl group. The RNA is purified by ethanol precipitation prior to proceeding to library preparation.

Total RNA is reverse-transcribed to cDNA. For labeled RNA transcripts, reverse transcriptase incorporates a G instead of an A at positions where reduced *S4U-modified nucleotides are encountered.
Second-strand cDNA synthesis is then performed to generate a double-stranded SLAMseq RNA-Seq library.

PCR is used to amplify the SLAMseq RNA-Seq library and to add indices and full adapter sequences for next-generation sequencing. Sequencing reads with T>C mutations distinguish labeled from unlabeled transcripts, which are read-out during data analysis.


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Ordering Information

Cat. №Product Name
061.24SLAMseq Kinetics Kit - Anabolic Kinetics Module, 24 preps
229.24SLAMseq Anabolic Kinetic 3’ mRNA-Seq with QuantSeq FWD V2, 24 preps

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