Cell type-specific transcriptome analysis is an essential tool in understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel in vivo metabolic RNA sequencing method, SLAM-ITseq, which metabolically labels RNA with 4-thiouracil in a specific cell type in vivo followed by detection through an RNA-seq-based method that specifically distinguishes the thiolated uridine by base conversion. This method has successfully identified the cell type-specific transcriptome in three different tissues: endothelial cells in brain, epithelial cells in intestine, and adipocytes in white adipose tissue. Since this method does not require isolation of cells or RNA prior to the transcriptomic analysis, SLAM-ITseq provides an easy yet accurate snapshot of the transcriptional state in vivo.

Features SLAMseq Metabolic RNA Labeling Kit for RNA-Seq and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. Here we combine SLAM-seq, a method for direct quantification of newly synthesized mRNAs, with pharmacological and chemical-genetic perturbation to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETi). We find that BRD4 acts as general co-activator of RNA polymerase II (Pol2)-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETi deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de-novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

Features SLAMseq Metabolic RNA Labeling Kit for RNA-Seq and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

The combination of metabolic RNA labeling with biochemical nucleoside conversion now adds a broadly applicable temporal dimension to RNA sequencing.

Features SLAMseq Metabolic RNA Labeling Kit for RNA-Seq

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s⁴U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N⁶-methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina and SLAMseq Metabolic RNA Labeling Kit for RNA-Seq