ID: 1518171
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA526995 | ENA |
REPOSITORIES: ENA
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Project description:Eukaryotic genes often generate a variety of RNA isoforms that can lead to functionally distinct protein variants. The synthesis and stability of RNA isoforms is however poorly characterized. The reason for this is that current methods to quantify RNA metabolism use short-read sequencing that cannot detect RNA isoforms. Here we present nanopore sequencing-based Isoform Dynamics (nano-ID), a method that detects newly synthesized RNA isoforms and monitors isoform metabolism. nano-ID combines metabolic RNA labeling, long-read nanopore sequencing of native RNA molecules and machine learning. nano-ID derived RNA stability estimates enable a distinctive evaluation of stability determining factors such as sequence, poly(A)-tail length, RNA secondary structure, translation efficiency and RNA binding proteins. Application of nano-ID to the heat shock response in human cells reveals that many RNA isoforms change their stability. nano-ID also shows that the metabolism of individual RNA isoforms differs strongly from that estimated for the combined RNA signal at a specific gene locus. nano-ID enables studies of RNA metabolism on the level of single RNA molecules and isoforms in different cell states and conditions.
2020-05-06 | GSE127890 | GEO
Project description:Stem cell functions require activation of stem cell-intrinsic transcriptional programs as well as intimate extracellular interactions with a niche microenvironment. How the core pluripotency transcriptional machinery controls residency of stem cells in the niche microenvironment is unknown. Here we show that the helix loop helix transcriptional regulators Id (Inhibitors of DNA binding) are the master regulators that coordinate stem cell activities with anchorage of neural stem cells (NSCs) to the embryonic and postnatal niche. Conditional inactivation of Id genes (Id1, Id2 and Id3) in the mouse NSC compartment triggered detachment of embryonic and post-natal NSCs from the ventricular and vascular niche respectively, followed by premature differentiation. Through an unbiased interrogation of the gene modules directly targeted by deletion of Id genes in NSCs, we discovered that Id proteins repress the bHLH-mediated activation of Rap1GAP, thus serving to maintain the GTPase activity of RAP1, a key mediator of cell adhesion. Preventing the elevation of Rap1GAP efficiently countered the consequences of Id loss on NSC-niche interaction and stem cell identity. Thus, by preserving anchorage to the extracellular environment of NSCs, Id activity synchronizes NSC functions to residency in the specialized niche. We generated Id-cTKO mice carrying a Cre-recombinase-oestrogen-receptor-T2 (Cre-ER) allele targeted to the ubiquitously expressed ROSA26 locus (Id-cTKO-Rosa-Cre-ER). In this system, 4-hydroxytamoxifen (4-OHT) releases the Cre recombinase inhibition and allows recombination of genomic loxP sites. Indeed, efficient deletion of Id1 and Id2 with complete loss of Id protein expression was detectable after treatment of NSCs with 4-OHT for 72 h. Total RNA was extracted from triplicate samples of Id-cTKO-Rosa-Cre-ER NSCs treated for different times (6 h, 12 h, 18 h, 24 h, 48 h, 96 h, 144 h) with 4-OHT or control vehicle and used for analysis on Illumina MouseWG-6 expression BeadChip. The raw array data was normalized using the Bioconductor package Lumi using quantile normalization.
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Project description:Purpose: The goal of this study is to compare endothelial small RNA transcriptome to identify the target of OASL under basal or stimulated conditions by utilizing miRNA-seq. Methods: Endothelial miRNA profilies of siCTL or siOASL transfected HUVECs were generated by illumina sequencing method, in duplicate. After sequencing, the raw sequence reads are filtered based on quality. The adapter sequences are also trimmed off the raw sequence reads. rRNA removed reads are sequentially aligned to reference genome (GRCh38) and miRNA prediction is performed by miRDeep2. Results: We identified known miRNA in species (miRDeep2) in the HUVECs transfected with siCTL or siOASL. The expression profile of mature miRNA is used to analyze differentially expressed miRNA(DE miRNA). Conclusions: Our study represents the first analysis of endothelial miRNA profiles affected by OASL knockdown with biologic replicates.
2022-09-06 | GSE186354 | GEO
Project description:Mammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs and looping interactions. Currently, there is a great need to evaluate the link between chromatin topology and genome function across many biological conditions and genetic perturbations. Hi-C generates high quality, high resolution maps of looping interactions genome-wide, but is intractable for high-throughput screening of loops across conditions due to the requirement of an enormous number of reads (>6 Billion) per library. Here, we describe 5C-ID, an updated version of Chromosome-Conformation-Capture-Carbon-Copy (5C) with restriction digest and ligation performed in the nucleus (in situ ChromosomeConformation-Capture (3C)) and ligation-mediated amplification performed with a new double alternating design. 5C-ID reduces spatial noise and enables higher resolution 3D genome folding maps than canonical 5C, allowing for a marked improvement in sensitivity and specificity of loop detection. 5C-ID enables the creation of high-resolution, high-coverage maps of chromatin loops in up to a 30 Megabase subset of the genome at a fraction of the cost of Hi-C.
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