SLAMseq RNA Kinetics Kits

SLAMseq: High-Throughput Metabolic Sequencing of RNA

Analyze transcriptome-wide kinetics of RNA synthesis and turnover
Measure nascent RNA expression and transcript stability
Enhance the temporal resolution of differential expression
Identify primary and secondary transcriptional targets
No pull-down or biochemical isolation required
Use in combination with QuantSeq 3’ mRNA-Seq for cost-effective,
high-throughput metabolic sequencing

SLAMseq: Metabolic RNA Labeling

This product can be used for SARS-CoV-2 research.

SLAMseq is a high-sensitivity method for time-resolved measurement of newly synthesized and existing RNA in cultured cells. SLAMseq enables resolution of RNA synthesis and degradation kinetics.

Lexogen offers a family of kits based on the new SLAMseq method: Thiol (SH)-Linked Alkylation for the Metabolic sequencing of RNA. SLAMseq enables the identification and quantification of newly synthesized (nascent) and existing RNA from the same sample in parallel, without the need for biochemical isolation. SLAMseq can be readily applied to living cell experiments. Combined with QuantSeq 3’ mRNA-Seq library preparation, SLAMseq provides a complete user friendly and high-throughput solution for analyzing transcriptome-wide RNA synthesis and turnover kinetics.

The SLAMseq kits portfolio includes Explorer and Kinetics Modules, which cover the entire metabolic RNA labeling experiment, from optimizing labeling conditions, to labeling of nascent or existing RNA and alkylation for downstream NGS library preparation.

Optimize 4-Thiouridine Labeling Conditions

The SLAMseq Explorer Kits facilitate the optimization of RNA labeling experiments using 4-Thiouridine (S4U). Assess cell viability and determine optimal concentrations for specific cell types and experimental durations using the SLAMseq Explorer Kit – Cell Viability Titration Module (Cat. No. 059).

Assess Nascent RNA Levels and RNA Synthesis Rates

Distinguish newly-synthesized (nascent) RNA from total RNA using the SLAMseq Kinetics Kit – Anabolic Kinetics Module (Cat. No. 061). 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.

Identify Direct Transcriptional Targets of Any Gene

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

Measure Global S4U Uptake under Experimental Conditions

S4U incorporation rates may vary for different cell types and culture methods. The SLAMseq Explorer Kit – S4U Incorporation Module (Cat. No. 060) can be used to measure the efficiency of S4U incorporation into newly synthesized RNA.

Monitor RNA Degradation Rates

The SLAMseq Kinetics Kit – Catabolic Kinetics Module (Cat. No. 062) can be used to measure transcript degradation rates. Cells are first grown in S4U-containing medium for a specified time period to label existing RNA. S4U is then replaced by unmodified uridine (U) and RNA is sampled. RNA degradation rates are calculated from the decrease in S4U-labeled RNA over time.

Analyze Transcriptome-Wide Expression Dynamics

SLAMseq resolves transcript expression dynamics on a transcriptome-wide scale. Individual transcript RNA synthesis and degradation rates can be measured directly.

SLAMseq Kit Selection Guide


Cat. No	Kit Type	Application
059.24	SLAMseq Explorer Kit – Cell Viability Titration Module	Assess S4U toxicity in target cell lines. Optimize S4U labeling concentrations.
060.24	SLAMseq Explorer Kit – S4U Incorporation Module	Measure S4U uptake and incorporation rates using HPLC analysis.
061.24	SLAMseq Kinetics Kit – Anabolic Kinetics Module	Label newly transcribed RNA with S4U. Measure nascent RNA expression. Analyze RNA synthesis kinetics.
062.24	SLAMseq Kinetics Kit – Catabolic Kinetics Module	Label existing RNA with S4U. Assess transcript stability. Analyze RNA degradation kinetics.

RNA Sequencing

Combining SLAMseq with Lexogen’s QuantSeq 3’ mRNA-Seq or QuantSeq Flex Targeted RNA-Seq Kits, provides a complete user-friendly solution for high-throughput metabolic RNA sequencing experiments.

Total RNA from SLAMseq experiments can be used directly for the QuantSeq protocol. Simply isolate total RNA, alkylate with iodoacetamide, prepare libraries, and sequence. No additional pull-down of labeled RNA is required!

The QuantSeq mRNA-Seq 3’ tag-based approach features a streamlined workflow that enables time- and cost-efficient library preparation. QuantSeq generates stranded libraries that require lower read depths than standard RNA-Seq, thus enabling samples from multiple timepoints and in replicates to be multiplexed in a single sequencing lane or run. Or use QuantSeq Flex with custom primers for first or second strand synthesis, or both, to generate targeted ready-to-sequence libraries.

Step1:

Total time for Step:

Hands-on time for Step :

Cultured cells are grown in 4-Thiouridine (S4U) to allow S4U nucleotides to be incorporated into newly synthesized RNA. The labeling duration depends on the type of experiment.

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

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.

Stefan L. Ameres from the Institute of Molecular Biotechnology (IMBA) in Austria gave a talk “SLAMseq: Thiol-linked alkylation for the metabolic sequencing of RNA” at Advances in Genome Biology and Technology (AGBT) General Meeting in Orlando, Florida (February 15, 2018).

Frequently Asked Questions

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Please also check our General Guidelines and FAQ resources!

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User Guide – update 06.12.2022

Application Note – update 23.05.2023

Product Flyer for Drug Development – update 24.05.2023

Safety Data Sheet

Request Safety Data Sheet

If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.

We recommend the use of the SLAMdunk analysis pipeline for analyzing SLAMseq sequencing data, as used by Herzog et al., Thiol-linked alkylation of RNA to assess expression dynamics (Nature Methods, 2017: DOI:10.1038/nmeth.4435). This modified alignment algorithm is specially adapted to report read counts for T>C containing reads for downstream analysis.

For further details of SLAMseq data analysis, please contact support@lexogen.com.

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SLAMseq: High-Throughput Metabolic Sequencing of RNA

Description

SLAMseq: Metabolic RNA Labeling

Optimize 4-Thiouridine Labeling Conditions

Assess Nascent RNA Levels and RNA Synthesis Rates

Identify Direct Transcriptional Targets of Any Gene