Expression profiling by high throughput sequencing Other
Summary
Promoters and enhancers are key cis-regulatory elements, but how they operate to generate cell-type-specific transcriptomes is not fully understood. We developed a simple and robust approach to sensitively detect 5’-ends of nascent RNAs (NET-CAGE) in diverse cells and tissues, including unstable transcripts such as enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site (TSS) level, characterizing the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without the influence of RNA turnover, and show that enhancer-promoter pairs are generally activated simultaneously upon stimulation. By integrating NET-CAGE data with chromatin interaction maps, we show that cis-regulatory elements are topologically connected according to their cell-type specificity. We identified new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our NET-CAGE dataset derived from human and mouse cells expands the FANTOM5 catalogue of transcribed enhancers, with broad applicability to biomedical research.
Overall design
Our study consists of 144 samples (NET-CAGE: 87 samples, CAGE: 43 samples and RNA-seq: 14 samples). Five ENCODE human cell lines and 2 mouse tissues were used to generate these data sets. A) NET-CAGE samples for 5 human cell lines: MCF-7 (46 samples: 24 samples for time course experiment which consists of 8 time points with 3 biological replicates for each time point, 6 samples with different cell freezing conditions with 2 biological replicates for each condition, and 16 samples for 6 urea treatment conditions with 2-6 biological replicates for each condition), GM12878 (14 samples for 6 urea treatment conditions with 2-4 biological replicates for each condition), HeLa-S3, HepG2, K562 (6 samples for each cell line, comprising of 3 urea treatment conditions with 2 biological replicates for each condition). B) CAGE samples for 5 human cell lines: MCF-7 (26 samples: 24 samples for time course experiment which consists of 8 time points with 3 biological replicates for each time point, and 2 biological replicates without any treatment), GM12878, HeLa-S3, HepG2, K562 (2 biological replicates for each cell line). C) NET-CAGE samples for mouse tissues: brain (1 sample with 3 technical replicates), kidney (2 biological replicates with 3 technical replicates). D) CAGE samples for mouse tissues: brain (1 sample with 3 technical replicates), kidney (2 biological replicates with 3 technical replicates). E) Nascent RNA-seq samples for MCF-7 (3 urea treatment conditions with 2 biological replicates for each condition). F) Total RNA-seq samples for MCF-7 (2 biological replicates). G) Nascent RNA-seq samples for mouse tissues: brain (1 sample), kidney (2 biological replicates). H) Total RNA-seq samples for mouse tissues: brain (1 sample), kidney (2 biological replicates).
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