SLAMseq: High-Throughput Metabolic Sequencing of RNA
2Pages

LESODEn Enabling complete transcriptome sequencing 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 • No pull-down or biochemical isolation required • Use in combination with QuantSeq 3' mRNA-Seq for cost-effective, high-throughput metabolic sequencing Introduction - metabolic Sequencing of RnR Metabolic RnR sequencing combines labeling of newly synthesized RnR transcripts with RnR-Seq readout. Existing approaches utilize nucleotide derivatives such as 4-Thiouridine and biochemical pulldown to separate nascent and existing RnR for library preparation R These protocols are typically cost-, time-, and labor-intensive, require high amounts of RnR input, and often produce low signal quality 5 SLfimseq Technology Lexogen now offers a family of kits based on a new transcriptomewide, quantitative, fast, and reliable method: SLAMseq (thiol (SH)-Linked Alkylation for the Metabolic Sequencing of RnR) 2. SLAMseq uses 4-Thiouridine (S4U) to label nascent RnR in cultured cells (Fig. 1). The key feature of the workflow is an alkylation step, which uses iodoacetamide (IRA) to modify S4U nucleotides, leading to nucleotide conversion during reverse transcription. This results in thymine-to-cytosine (T>C) mutations in sequencing reads from S4U-labeled transcripts. Bioinformatic analysis of T>C-containing read counts can then be used to analyze nascent RnR levels. SLRrnseq Kits Lexogen's SLAMseq kits provide a complete solution for metabolic RnR-Seq experiments, right through from optimizing S4U labeling conditions, to performing S4U labeling kinetics experiments, and subsequent RnR isolation and alkylation (Table 1). Table 1 | Lexogen's SLAmseq kits. l S4U o^o o°o Cultured Cells Figure 1 | The SLAmseq workflow. Cultured cells are treated with 4-Thiouridine (S4U) for labeling of nascent RnR (green). Total RnR is purified, and alkylation of the 4-thiol group is induced by the addition of iodoacetamide (IRA). During library preparation, shown here using the QuantSeq 3' mRnA-Seq Library Prep Kit, the presence of the resulting carboxyamidomethyl-group causes reverse transcriptase to incorporate guanine (G, in red) instead of adenine (A, in black) at any position where a reduced *S4U-modified nucleotide is encountered. In this way, nascent RnR can be distinguished from existing RnR by the presence of T>C mutations (red stars) during subsequent data analysis. Ordering Information Catalog numbers: 059 (SLAMseq Explorer Kit - Cell Viability Titration Module) 060 (SLAMseq Explorer Kit - S4U Incorporation Module) 061 (SLAMseq Kinetics Kit - Anabolic Kinetics Module) 062 (SLAMseq Kinetics Kit - Catabolic Kinetics Module) 063 (SLAMdunk Data Analysis for SLAMseq Integrated on Bluebee Find more about SLAmseq at www.lexogenx°m

By defining nascent and total RNA levels for each sample, SLAMseq extends the depth of information provided by differential expression experiments. To demonstrate this, an anabolic kinetics experiment was conducted, using a 2-hour (120 minute) S4U labeling duration, comparing two treatment conditions to untreated controls at each timepoint (Table 2). Table 2 | SLAMseq kinetics experiment for differential expression. Increasing strength treatment series (T1, T2) and control (no treatment) with S4U labeling (12 sample groups, numbered 1-12). To assess the maximal effect of treatment,...