Acrylonitrile-mediated nascent RNA sequencing for transcriptome-wide profiling of RNA dynamics
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA508892 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8290740_1.fastq.gz | Fastqsanger.gz | |||
| SRR8290740_2.fastq.gz | Fastqsanger.gz | |||
| SRR8290750_1.fastq.gz | Fastqsanger.gz | |||
| SRR8290750_2.fastq.gz | Fastqsanger.gz | |||
| SRR8290741_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 28 |
Similar Datasets
Project description:RNA sequencing has greatly facilitated gene expression studies but are weak in studying temporal RNA dynamics. This issue can be addressed by profiling nascent RNAs. Here, we presented a NGS-compatible method for nascent RNA analysis in single-base resolution based on s4U cyanoethylation reaction. Our strategy can high-efficiently recover nascent RNA information from total RNAs. We validated our method by measuring RNA stabilities transcriptome wide in human cells. By capturing immediate gene expression changes, we successfully determined the direct gene targets of a G-quadruplex stabilizer, which has been regarded as potential antitumor drug. We also identified the influence of G-quadruplex stabilization to mRNA maturation. Furthermore, we found G-quadruplex stabilizer positively regulated the transcriptional activity of upstream antisense RNAs, uncovering its novel potential gene regulation pathway in vivo.
2020-06-19 | GSE123483 | GEO
Project description:RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile-mediated uridine-to-cytidine (U-to-C) conversion (AMUC-seq) via 4-thiouridine (s4U) cyanoethylation is presented. This method converts s4U base-pairing with guanine through the nucleophilic addition of s4U to acrylonitrile. The high reaction efficiency permits AMUC-seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC-seq is validated by being used to detect mRNA half-lives and investigating the direct gene targets of a G-quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC-seq also confirms G-quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC-seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription-modulating drugs.
| S-EPMC7175251 | biostudies-literature
Project description:Revealing nascent RNA processing dynamics with nano-COP
| PRJNA645078 | ENA
Project description:Dynamic imaging of nascent RNA reveals general principles of transcription dynamics and pervasive recursive splicing [nascent RNA profiling]
| PRJNA555414 | ENA
Project description:The coupled activities of RNA polymerase and the spliceosome are responsible for the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex further reveals widespread splice site selection within introns. We propose and validate a unified theoretical model for transcription and splicing based on pervasive stochastic recursive splicing.
2021-05-13 | GSE134392 | GEO
Project description:To address co-transcriptional pre-mRNA processing events, Saccharomyces nascent RNA was isolated by chromatin fractionation and analyzed on a genome-wide high density tiling microarray. Co-transcriptional splicing efficiencies were derived by determination of intronic relative to exonic sequence concentrations. Nascent RNA, mRNA and genomic DNA were isolated from wild-type Saccharomyces cells and analyzed on a genome-wide high-density tiling microarry (Saccharomyces Tiling 1.0R Array, Affymetrix). At least 3 independent biological replicates were analyzed (nascent RNA (nRNA): 3 replicates, mRNA: 3 replicates, genomic DNA (gDNA): 4 replicates).
2010-11-26 | E-GEOD-24040 | biostudies-arrayexpress
Project description:During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3’-end cleavage, and polyadenylation. Here, we describe nanopore analysis of CO-transcriptional Processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing (NET-seq), which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3’ ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read–based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 days.
2021-01-06 | GSE154079 | GEO
Project description:To capture chromatin dissociation dynamics of nascent RNA transcripts we labeled MCF-7 cells with 4-thio-uridine (4-SU) for 8 minutes, then chased with an excess of uridine for 5, 10, 15 and 20 min. At each pulse-chase time point we performed TT-seq from the chromatin-associated and nucleoplasmic fraction. The ratios of spike-in normalized chromatin-associated to chromatin-released nascent RNA read coverage over genomic features (transcripts' last exons) were fitted on an exponential decay function to extract an estimated chromatin association half-live time for each transcript.
2023-09-21 | GSE218726 | GEO
Project description:RNA sequences are expected to be identical to their corresponding DNA sequences. Advances in technologies have enabled deep sequencing of nucleic acids that uncovered exceptions to the one-to-one relationship between DNA and RNA sequences. Previously in human cells, post-transcriptional RNA editing was the only known mechanism that changes RNA sequences from the underlying DNA sequences. Here, we sequenced nascent RNA and found all 12 types of RNA-DNA differences. Using various experimental analyses, we validated this finding. Our results showed that sequences of nascent RNAs within 40 nucleotides of the exit channel of RNA polymerase II already differ from the corresponding DNA sequences. These RNA-DNA differences are mediated by RNA processing steps closely coupled with transcription and not by known deaminase-mediated RNA editing mechanisms nor during NTP incorporation by Pol II. This finding identifies sequence substitution as part of co-transcriptional RNA processing. We sequenced nascent RNA using global run-on sequencing, GRO-seq from human B-cells from two individuals and a variant of the GRO-seq procedure, known as precision run-on sequencing, PRO-seq. The RNAs are prepared after a short run-on assay performed with isolated nuclei in the presence of Br-UTP. The isolated RNAs are base hydrolyzed to ~100 nucleotides and affinity purified with anti-BrU beads three times at each successive step of preparing the RNAs for orientation-specific sequencing using Illumina technology. The 5M-bM-^@M-^Y ~half of each sequence represents nascent RNA made in the cell and the 3M-bM-^@M-^Y ~half represents sequences made in vitro during the run-on reaction. The precision variation, PRO-seq, incorporates one or at most a few biotin-labeled nucleoside triphosphates during the run-on, and sequencing from the 3M-bM-^@M-^Y end of this affinity purified, nascent RNA maps the cellular location of engaged polymerases with near single nucleotide precision. We obtained ~ 100 million 100-nucleotide uniquely mapped GRO-seq reads from B-cells of two individuals. For one subject, we also carried out pGRO-seq and obtained 60 million uniquely mapped reads. In addition, we sequenced ~135 million uniquely mapped RNA-seq reads, and the corresponding DNA of the two individuals to 30X and 60X coverage. Additionally, we isolated and sequenced nascent RNA with an alternate method described by Wuarin and Schibler (1994) in order to compare chromatin-bound RNA to the very nascent RNA from PRO-seq. We obtained ~190 million uniquely mapped reads from chormatin-bound RNA-seq.
2014-02-21 | E-GEOD-39878 | biostudies-arrayexpress