# TT-seq ## expand\_more ## Method Description TT-seq is a variant of 4-thiouridine (4sU) sequencing. This approach labels newly synthesized RNA by metabolic incorporation of 4sU in live cells. Analysis of this RNA can provide information on RNA synthesis, co-transcriptional processing, and degradation (depending on experimental design). ![TTseq diag](/sites/g/files/omnuum3651/files/styles/hwp_1_1__720x720_scale/public/txcore/files/ttseq-diagram-web.png?itok=ly16MKZf) ## Applications - Time resolved measurement of RNA output - Sensitive differential expression analysis - Captures unstable RNA species, such as introns and non-coding RNAs ## Service Description Users submit total RNA isolated from cells pulsed with 4sU. The core fragments the total RNA, enriches 4sU labeled fragments, constructs Illumina compatible RNA-seq libraries, and performs preliminary data analysis. The amount of material required depends on duration of metabolic labeling and the cell type being labeled and can range from 0.5-2% of total RNA. We request that users provide 50-100ug of total RNA but please contact us for discussion of the amount of labeled RNA required to construct libraries for your project. ## Sample Preparation ### Required Inputs Sort**Method****Minimum # cells** **Requested # of cells** **Cell condition** **Spike-in provided** **Spike-in required** TT-seq 5-8 million \* (≥50 ug total RNA) 5-8 million\* (50-100 ug total RNA) 4sU labeled cell lysate in Trizol *OR* 4sU labeled total RNA yes yes \* The required number of cells needed to enrich sufficient 4sU-labeled RNA for library construction depends on the cell line being labeled and the duration of labeling. Shorter labeling times and cells with low yields of RNA per cell will require larger inputs. The cell counts above are estimated for HEK293T cells labeled 10-20 minutes with 4sU. ### 4sU Labeling Protocol We suggest labeling and harvesting cells according to our [**4sU labeling protocol**](/sites/g/files/omnuum3651/files/2025-06/NTC%204sU%20Labeling%20and%20Harvest%20Protocol%20v4%20PROD.docx "4sU Labeling Protocol") . Efficient labeling requires cells that are growing well and well below confluent density. Our protocol contains general guidance but please feel free to reach out to us with questions. ### Sample Spiking We accept TTseq samples submitted as isolated total RNA (preferred) or trizol lysate. The decision depends on how the samples need to be spiked and, to a lesser extent, the user's experience working with RNA. We can advise which method is suitable for your project. RNA samples Technical Spiking (default) Biological Spiking ## Technical Spiking (default) Users submit 4sU labeled total RNA and we spike in 4sU labeled RNA (from Drosophila S2 cells; prepared by NTC) proportional to the **amount (ug) of RNA**. This serves as a technical control for 4sU enrichment and library preparation (akin to ERCC spiking in bulk RNAseq). Technical spikes are useful where experimental conditions affect subsets of the transcriptome not the majority of transcripts per cell. - Good for depletion of specific transcription factors, control vs drug, etc. - Does not capture global changes in transcription in cases such as depletion of global transcription factors, RNA degradation machinery, cell differentiation time courses, etc. In cases where global alterations in transcription between conditions are expected, biological spiking is recommended. We highly recommend using our suggested [**RNA isolation protocol**](/sites/g/files/omnuum3651/files/2025-08/NTC%20Modified%20miRNeasy%204sU%20RNA%20isolation%20PROD.docx "4sU RNA Isolation"). RNA precipitated directly from the aqueous phase of dense trizol lysates can easily be contaminated by trace organics and proteins including endogenous RNases. This column based cleanup yields high quality RNA and allows in-line DNase treatment to further reduce background. ## Biological Spiking Samples are submitted as cell lysates in trizol and spiked with 4sU labeled cell lysate (from Drosophila S2 cells; prepared by NTC) proportional to the **number of cells** reported to us. Biological spikes capture changes in RNA content per cell and are the only way to normalize data when global, unidirectional changes in RNA per cell occur such a drug that globally suppresses transcription where the goal is to identify transcripts altered relative to this shifted baseline. - Good for all biological comparisons. - More technically challenging than technical spiking with critical steps performed by the user prior to submission. - **Accurate cell counts are CRITICAL** - Labeling and cell harvest must be performed with great care. Because NTC cannot verify cell counts in lysates users are *entirely responsible for providing accurate counts*. The core takes no responsibility for incorrect normalization (and potentially misleading results) arising from inconsistent 4sU labeling or inaccurate cell counts. - **Each sample lysate must fit in a single 1.5 or 2 mL low binding tube** - DO NOT OVERFILL TUBES. Allow head-space for liquid expansion upon freezing. It is impractical for us to process lysate volumes greater than 2mL. - Do not divide lysate into multiple tubes as cell material may be unequally distributed skewing the ratio of cells per mL lysate which must remain consistent for accurate spiking. - **Homogenize cells completely into trizol** - Clumps of cell debris can cause errors during aliquotting and handling. - **Users are responsible for safe packing and shipping of trizol** - NTC may decline to process leaking or broken tubes. ## Further Reading **Schwalb, Björn, Margaux Michel, Benedikt Zacher, Katja Frühauf, Carina Demel, Achim Tresch, Julien Gagneur, and Patrick Cramer**. TT-Seq Maps the Human Transient Transcriptome. *Science* 352, no. 6290 (June 3, 2016): 1225–28. PMID: 27257258 .